BACKGROUND
among heritable factors that influence phenotypic expression are sequence polymorphisms in genic regions that affect gene expression rather than protein structure  <cit> .
the influence of sequence variation linked to the gene sequence on the regulation of gene expression  has been studied experimentally in h. sapiens at single genes for decades  <cit> , and, more recently, in various multi-gene approaches in s. cerevisiae  <cit> , s. purpuratus  <cit> , d. melanogaster and d. simulans  <cit> , m. musculus  <cit> , z. mays  <cit> , and h. sapiens  <cit> .
in studies with human tissues, these efforts have characterized cis sequence effects on gene expression as common and heritable  <cit>  and have used both unrelated and related individuals to quantify such cis sequence effects  <cit> .
array-based genotyping and gene expression platforms  <cit>  have been essential for multi-gene approaches, and to generate data enabling investigation of the potential effect of sequence variation not linked to the gene on gene expression .
we used previously generated genomic resequencing data and, for this study, quantified in vitro transcript levels from thirty unrelated individuals at several hundred candidate genes commonly studied in cancer research.
we identified a subset of candidate genes with abundant sequence and gene expression variation.
we evaluated potential cis sequence effects using individual single nucleotide polymorphisms , all snps at a candidate gene considered jointly and haplotype phylogenies and diplotypes.
we compared our findings to the published cis sequence effects literature and to the existing gene expression regulation literature available for those candidate genes that exhibited cis sequence effects.
RESULTS
gene expression data quality
thirty lymphoblastoid cell lines drawn from the snp500cancer resource were cultured in triplicate and total rna extracted .
gene expression profiling was performed on the n =  <dig> samples using a custom illumina sentrix® array matrix- <dig> microarrays containing  <dig> mer probes targeting  <dig> genes relevant to cancer research .
gene expression data from one array were excluded from further analysis due to a within individual cell line linear r <dig> correlation for all genes of <95%, while all remaining within individual cell line correlations were ~99%.
this correlation statistic reflects variation at all levels of the experiment: cell culture, rna extraction, rna labeling, and array performance.
based on these high quality data, further analysis used normalized mean gene expression signal data from the three replicate arrays.
selection of genes for analysis of potential cis effects
we used two threshold criteria to select genes from the n =  <dig> genes for further analysis: a signal amplitude of ≥ <dig> normalized units ; and a between cell line coefficient of variation  of ≥20% .
these criteria identified n =  <dig> genes with sufficient, and sufficiently variable, gene expression for further analysis.
we used the criterion of ≥ <dig> snps per candidate gene in the n =  <dig> dnas to identify n =  <dig> candidate genes with sufficient sequence variation for analysis.
we then compared the n =  <dig> genes with sufficient gene expression variance and n =  <dig> genes with sequence variation derived from the snp500cancer resequencing program, a component of the cancer genome anatomy project of the national cancer institute  <cit>  and identified a subset of n =  <dig> genes  for the analysis of potential cis sequence effects .
gene expression of selected genes
the mean  rank-invariant normalized gene expression signal of the subset of n =  <dig> genes was  <dig>  units , the mean  intra cell line replicate culture correlation coefficient was  <dig>  , and the mean  between cell line coefficient of variation  was  <dig>  .
mean noise  was  <dig> units +/-  <dig> units.
pcna had the largest mean gene expression, tp73l had the smallest gene expression and the largest cv, and pten had the smallest cv.
selection of snps at genes for association analysis
the number  of polymorphic snps per gene ranged from  <dig> to  <dig>  and the number of tag  and singleton snps per gene ranged from  <dig> to  <dig>  in the group of n =  <dig> genes selected for association analysis .
two genes  were excluded from further analysis as there were no snps at these genes with minor allele frequencies >5%.
two snps with genotype completion  rates of 63% and 77% were excluded.
after these exclusions, there were n =  <dig> tag and singleton snps at  <dig> genes available for analysis with a mean  genotype completion rate of  <dig> %  .
the distribution of snp genotypes in the n =  <dig> cell lines was evaluated for hardy weinberg equilibrium using asymptotic and exact tests; there were no snps exhibiting exact test p values <  <dig> , and two exhibiting exact test p values <  <dig> , both were at tp73l.
the distribution of the flanking, untranslated region, coding and intronic snps was 27%, 15%, 12% and 46%, respectively.
four genes  had only one informative tag or singleton snp and were excluded from haplotype-based analyses, leaving n =  <dig> genes with a range of  <dig> to  <dig> tag and singleton snps for haplotype based analyses.
the gene with the largest number of tag and singleton snps  generated too many terms for the implementation of the sam model to compute.
the remaining n =  <dig> genes, comprised of n =  <dig> tag and singleton snps , have results from all snp and haplotype based methods.
the number of snps before and after selection of tag and singleton snps were uncorrelated  with mean normalized signal, the standard deviation, intra cell line correlation coefficient, or inter cell line coefficient .
*there was one snp with maf > 5% at these two genes; tag snp analysis was not performed.
the single snp with maf > 5% at each gene was used for snp-based analyses.
significant cis sequence effects identified via three association methods
n =  <dig> individual snps at six genes were significantly associated with gene expression signal, three genes exhibited significant association to gene expression in the single additive model, and three genes exhibited one or more statistically significant haplotype partitions .
the distribution of flanking, untranslated region, coding and intronic snps significantly associated to gene expression  was not significantly different from the distribution of snps not exhibiting association to gene expression .
three  of the six genes identified using individual snps were identified by one or both of the two gene-based association methods.
three genes  exhibit significant association in one or two methods and only a trend towards statistical significance in another method.
*statistically significant results are bolded.
**see additional file  <dig> for snp details.
snps in hybridization probes
we evaluated whether the  <dig> base pair oligonucleotide probe sequences for the eight genes that exhibited significant cis sequence effects exhibit sequence variation in the n =  <dig> individuals in this study .
there were two probes on the array for each gene, and all but two of the sequences  have been sequenced in the snp500cancer dnas.
two of the sixteen probe sequences exhibit variation in dbsnp , however neither exhibits variation in the n =  <dig> dna samples investigated here.
literature data on cis sequence effects at candidate genes in this study
we reviewed the multi-gene cis sequence effects literature that has investigated either allelic imbalance within individuals , generally defined as expression ratios ≥ <dig>  or ≤ <dig>  at transcribed heterozygous snps, or snp-wise linkage or association in related or unrelated samples of individuals, to gene expression  <cit> .
we compared results at the n =  <dig> genes studied in common by this study and at least one additional multi-gene cis sequence effects study  <cit>  .
to avoid issues of publication bias, we did not compare our results to the single gene cis sequence effects literature.
discussion
the proportion of genes exhibiting cis sequence effects
we identified nineteen publications that have investigated cis sequence effects in a multi-gene approach, one of which summarizes results from six other publications  <cit> .
these thirteen publications report results at an average of  <dig> genes .
the weighted average proportion of genes tested in these thirteen multi-gene studies considered by their authors to exhibit allelic imbalance or statistically significant cis sequence effects is  <dig> % .
we observe statistically significant cis sequence effects at eight of thirty genes  in our study, which is similar to that observed in the literature.
the proportion of gene expression attributable to cis sequence effects
the literature presents a variety of linkage and association techniques to estimate the proportion of gene expression variation accounted for by individual snps  <cit> , expressed either as an allelic ratio  <cit>  or as a proportion of variance explained  <cit> .
mean r <dig> estimates from n =  <dig>  <cit>  and n =  <dig>  <cit>  genes exhibiting significant cis sequence effects are 35% and 27%, respectively.
the average proportion  of gene expression variance explained by individually significant snps in the snp-wise regression analysis  in this study was 21% .
the average proportion of gene expression variance  explained by the most significant haplotype partitions at fcgr2b, lmo <dig> and pcna was 26% .
thus, current technical approaches suggest that approximately one-quarter to one-third of gene expression variation is attributable to cis sequence effects.
concordance of the multi-gene cis sequence effects literature and this study
pant et al.,  <dig>  examined eight genes for cis sequence effects in common with our study, the largest overlap in the extant literature .
both studies observed significant cis sequence effects at cyp1b <dig>  fcgr2b and mybl <dig>  pant et al., observed ai at tyms where we did not, while we observed cis sequence effects at lmo <dig> and nbs <dig>  where pant et al.
did not.
neither study observed cis sequence effects at jak <dig> and myc.
in toto, among the  <dig> genes jointly analyzed in this study and in the literature, and after excluding two genes with discordant literature results,  <dig> of  <dig> genes concordantly exhibit, and  <dig> of  <dig> genes concordantly do not exhibit, significant cis sequence effects, respectively.
concordance with results obtained using other experimental methods, as we have observed here, increases confidence in our results.
such evidence may provide the justification to proceed to more focused functional investigation of gene regulation.
the utility of different statistical methods used to evaluate cis sequence effects
we used three methods to test for the significance of cis sequence effects, after restricting the number of snps to tag and singleton snps at each gene.
regression of individual snps identified the largest number of genes exhibiting significant cis sequence effects and is the most commonly used method in the literature, after assessment of ai.
mander's single additive method   <cit>  and templeton's tree scanning method  <cit>  identified three genes; one gene, fcgr2b, was identified in common.
the latter two methods incorporate correction for multiple tests in their estimates of the significance of association.
rather than apply any formal correction for the multiple comparisons at a gene after regression analysis on snps, or after using multiple methods, we compare and contrast the results obtained from using the three methods.
differences between results obtained analyzing individual snps and the two methods that apply multiple test correction at the level of the gene suggests that much of the evidence for significant cis sequence effects in this sample of n =  <dig> lcls is too weak to survive multiple test correction, emphasizing the necessity to apply multiple test corrections to avoid elevated type i error  <cit> .
we also observed two examples of a gene exhibiting significant cis sequence effects with tree scanning, but not with the additive methods.
at lmo <dig>  the minor alleles of four snps define a haplotype partition that exhibits significantly reduced gene expression, and at pcna, the heterozygote diplotype exhibits significantly increased gene expression .
these latter findings depend upon the ability to model the effects of haplotypes within and between individuals.
we observed dominant effects for haplotypes at fcgr2b and lmo <dig> and heterotic effects for haplotypes at pcna, suggesting that searching for cis sequence effects using only additive model-based approaches may result in elevated type ii error rates.
biological relevance of cis sequence effects – fcgr2b as an example
the focus of this study is to evaluate association between variation in dna sequence and in vitro rna transcription in a group of candidate genes commonly studied in cancer research.
we briefly review some of the recent functional genomics literature for the candidate gene fcgr2b  and suggest below how a review of relevant genomic data and our cis sequence effects findings at fcgr2b might inform our understanding of this literature, as an example of how our findings might influence future fcgr2b snp association or functional analyses.
the snp500cancer program resequenced portions of ivs <dig>  exon  <dig>  ivs <dig>  ivs <dig> and exon  <dig> of fcgr2b, yielding n =  <dig> fcgr2b snps available with a minor allele frequency of ≥5% for analysis of gene expression variation.
after selecting one tag and five singleton snps to reduce the number of statistical tests performed with minimal loss of information  <cit> , significant cis sequence effects were observed at fcgr2b snp rs <dig>  .
the minor allele frequency of rs <dig> in our sample of n =  <dig> was 10%, similar to the minor allele frequencies of fcgr2b promoter and transmembrane snps previously studied, however, it should be noted that rs <dig> is a singleton snp, i.e., not strongly associated with the other snps available at fcgr2b.
linkage disequilibrium  within the fcgr2b genomic region in caucasian samples extends from the 3' end of ivs  <dig> distally in a punctate fashion, and there is some evidence for a separate 5' region of ld proximal to exon  <dig> .
rs <dig> has been genotyped by hinds et al.,  <dig> as afd <dig>  <cit>  with a minor allele frequency of 11%, and within their sample of n =  <dig> european american dnas, this snp exhibits strong ld with one additional snp .
however, both of these snps map to both intronic regions within the fcgr2b locus and also within the fcgr3b/fcgr3a intergenic region some  <dig> kbp proximal, suggesting that high sequence homology may be interfering with accurate map assignment.
there are two recent reports of copy number variation  in the region that are relevant: cnv of the fcgr3b locus is associated with nephritis in a rat model and in human patients  <cit> , and analysis of snp genotypes and genomic hybridization with the hapmap sample has identified a  <dig> kbp region as human copy number locus cnv_id_ <dig> containing the fcgr2a, hspa <dig>  fcgr3a, fcgr2b and fcrlm <dig> loci  <cit> .
our data at fcgr2b is concordant with data generated from both in vitro and ex vivo experimental strategies that sequence variation in the promoter  <cit>  and the coding region  <cit>  is associated with gene expression differences.
further, our data contributes to the evidence that a minor allele frequency of ~10% characterizes the snps that are associated with fcgr2b gene expression differences.
the inconsistent directionality of effect of the minor allele may be due to high sequence homology at the cnv_id_ <dig> locus affecting the physical and linkage disequilibrium mapping of the region, or may be due to incomplete linkage disequilibrium between the analyzed snp and a unanalyzed snp that may be causing the effect.
individuals wishing to investigate regulation of gene expression at fcgr2b in the future should include approaches necessary to characterize the physical, linkage disequilibrium, transcriptional and copy number architecture of the region.
strengths and limitations
strengths of this study include: high quality gene expression data from triplicate cell cultures for each lymphoblastoid cell line , with standardization of culture, rna extraction, labeling, amplification and hybridization conditions; the use of sequence-verified snps and the resequencing of nearly all expression array probe sequence regions; the use of multiple methods to evaluate evidence for significant cis sequence effects; and comparison of the results observed in this study to the published cis sequence effects literature at both the gene and snp levels.
limitations of this study include: the use of epstein-barr virus-transformed lcls; the modest number of lcls used; the association paradigm; and the absence of genetic assays that evaluate copy number in our sample.
limitations of lcls as reagents for the investigation of gene expression regulation include gene expression in a virally transformed surrogate ex vivo tissue, which may influence and potentially eliminate epstein-barr virus infection associated genes from gene expression investigation  <cit> .
systematic comparison of gene expression from lcls and non-surrogate, minimally processed tissues, e.g., peripheral blood lymphocytes, could be an approach towards validation of gene expression findings made in lcls  <cit> .
the modest number of cell lines used in this study limits statistical power, and is less than the number of cell lines used by some researchers  <cit> , however, the number of genes evaluated was also modest.
the association paradigm suffers from well known limitations  <cit> .
some publications testing large number of genes for cis sequence effects do not provide complete lists of genes tested or of genes exhibiting significant cis sequence effects on gene expression, therefore, we could not identify all genes studied in these reports.
also, due to the variety of approaches used in the literature, most comparisons are between categorical results of specific assays, i.e., it is generally not possible to compare quantitative data from different studies.
the mybl <dig> probe sequence regions were not resequenced in the n =  <dig> caucasian dna samples and thus the positive regression and treescan results at mybl <dig> could potentially be a false positive result due to an unidentified snp within the sequence complementary to these probes.
CONCLUSIONS
we tested for significant association between gene sequence variation and gene expression variation at n =  <dig> candidate genes in dna and rna from n =  <dig> lcls.
significant association between cis sequence and gene expression variation was observed in  <dig> out of  <dig> genes, and accounted for 26% of gene expression variation in three genes evaluated using an analysis of variance approach.
we utilized additive and analysis of variance  analytical approaches, and suggest that approaches that permit modeling of allelic effects may identify effects missed by additive models, although larger multi-gene studies would clarify the relative utility of the two approaches.
we reviewed the multi-gene cis sequence effects literature and found data on n =  <dig> of the candidate genes that we evaluated; most of that data is concordant with our results.
investigators using current technologies should expect to find cis sequence effects at about one quarter of candidate genes evaluated: these effects will explain about one quarter of gene expression variance.
snps associated with gene expression can be preferentially selected for genotyping and analysis in genetic association studies, or nominated for functional genomic investigations to characterize their role in the regulation of gene expression.

 among heritable factors that influence phenotypic expression are sequence polymorphisms in genic regions that affect gene expression rather than protein structure. the influence of sequence variation linked to the gene sequence on the regulation of gene expression has been studied experimentally in h. sapiens at single genes for decades, and, more recently, in various multi-gene approaches in s. cerevisiae, s. purpuratus, d. melanogaster and d. simulans, m. musculus, z. mays, and h. sapiens. in studies with human tissues, these efforts have characterized cis sequence effects on gene expression as common and heritable and have used both unrelated and related individuals to quantify such cis sequence effects. array-based genotyping and gene expression platforms have been essential for multi-gene approaches, and to generate data enabling investigation of the potential effect of sequence variation not linked to the gene on gene expression. we used previously generated genomic resequencing data and, for this study, quantified in vitro transcript levels from thirty unrelated individuals at several hundred candidate genes commonly studied in cancer research. we identified a subset of candidate genes with abundant sequence and gene expression variation. we evaluated potential cis sequence effects using individual single nucleotide polymorphisms, all snps at a candidate gene considered jointly and haplotype phylogenies and diplotypes. we compared our findings to the published cis sequence effects literature and to the existing gene expression regulation literature available for those candidate genes that exhibited cis sequence effects.  gene expression data quality thirty lymphoblastoid cell lines drawn from the snp500cancer resource were cultured in triplicate and total rna extracted. gene expression profiling was performed on the n = samples using a custom illumina sentrix® array matrix- microarrays containing mer probes targeting genes relevant to cancer research. gene expression data from one array were excluded from further analysis due to a within individual cell line linear r correlation for all genes of <95%, while all remaining within individual cell line correlations were ~99%. this correlation statistic reflects variation at all levels of the experiment: cell culture, rna extraction, rna labeling, and array performance. based on these high quality data, further analysis used normalized mean gene expression signal data from the three replicate arrays. selection of genes for analysis of potential cis effects we used two threshold criteria to select genes from the n = genes for further analysis: a signal amplitude of ≥ normalized units; and a between cell line coefficient of variation of ≥20%. these criteria identified n = genes with sufficient, and sufficiently variable, gene expression for further analysis. we used the criterion of ≥ snps per candidate gene in the n = dnas to identify n = candidate genes with sufficient sequence variation for analysis. we then compared the n = genes with sufficient gene expression variance and n = genes with sequence variation derived from the snp500cancer resequencing program, a component of the cancer genome anatomy project of the national cancer institute and identified a subset of n = genes for the analysis of potential cis sequence effects. gene expression of selected genes the mean rank-invariant normalized gene expression signal of the subset of n = genes was units, the mean intra cell line replicate culture correlation coefficient was, and the mean between cell line coefficient of variation was. mean noise was units +/- units. pcna had the largest mean gene expression, tp73l had the smallest gene expression and the largest cv, and pten had the smallest cv. selection of snps at genes for association analysis the number of polymorphic snps per gene ranged from to and the number of tag and singleton snps per gene ranged from to in the group of n = genes selected for association analysis. two genes were excluded from further analysis as there were no snps at these genes with minor allele frequencies >5%. two snps with genotype completion rates of 63% and 77% were excluded. after these exclusions, there were n = tag and singleton snps at genes available for analysis with a mean genotype completion rate of %. the distribution of snp genotypes in the n = cell lines was evaluated for hardy weinberg equilibrium using asymptotic and exact tests; there were no snps exhibiting exact test p values <, and two exhibiting exact test p values <, both were at tp73l. the distribution of the flanking, untranslated region, coding and intronic snps was 27%, 15%, 12% and 46%, respectively. four genes had only one informative tag or singleton snp and were excluded from haplotype-based analyses, leaving n = genes with a range of to tag and singleton snps for haplotype based analyses. the gene with the largest number of tag and singleton snps generated too many terms for the implementation of the sam model to compute. the remaining n = genes, comprised of n = tag and singleton snps, have results from all snp and haplotype based methods. the number of snps before and after selection of tag and singleton snps were uncorrelated with mean normalized signal, the standard deviation, intra cell line correlation coefficient, or inter cell line coefficient. *there was one snp with maf > 5% at these two genes; tag snp analysis was not performed. the single snp with maf > 5% at each gene was used for snp-based analyses. significant cis sequence effects identified via three association methods n = individual snps at six genes were significantly associated with gene expression signal, three genes exhibited significant association to gene expression in the single additive model, and three genes exhibited one or more statistically significant haplotype partitions. the distribution of flanking, untranslated region, coding and intronic snps significantly associated to gene expression was not significantly different from the distribution of snps not exhibiting association to gene expression. three of the six genes identified using individual snps were identified by one or both of the two gene-based association methods. three genes exhibit significant association in one or two methods and only a trend towards statistical significance in another method. *statistically significant results are bolded. **see additional file for snp details. snps in hybridization probes we evaluated whether the base pair oligonucleotide probe sequences for the eight genes that exhibited significant cis sequence effects exhibit sequence variation in the n = individuals in this study. there were two probes on the array for each gene, and all but two of the sequences have been sequenced in the snp500cancer dnas. two of the sixteen probe sequences exhibit variation in dbsnp, however neither exhibits variation in the n = dna samples investigated here. literature data on cis sequence effects at candidate genes in this study we reviewed the multi-gene cis sequence effects literature that has investigated either allelic imbalance within individuals, generally defined as expression ratios ≥ or ≤ at transcribed heterozygous snps, or snp-wise linkage or association in related or unrelated samples of individuals, to gene expression. we compared results at the n = genes studied in common by this study and at least one additional multi-gene cis sequence effects study. to avoid issues of publication bias, we did not compare our results to the single gene cis sequence effects literature. discussion the proportion of genes exhibiting cis sequence effects we identified nineteen publications that have investigated cis sequence effects in a multi-gene approach, one of which summarizes results from six other publications. these thirteen publications report results at an average of genes. the weighted average proportion of genes tested in these thirteen multi-gene studies considered by their authors to exhibit allelic imbalance or statistically significant cis sequence effects is %. we observe statistically significant cis sequence effects at eight of thirty genes in our study, which is similar to that observed in the literature. the proportion of gene expression attributable to cis sequence effects the literature presents a variety of linkage and association techniques to estimate the proportion of gene expression variation accounted for by individual snps, expressed either as an allelic ratio or as a proportion of variance explained. mean r estimates from n = and n = genes exhibiting significant cis sequence effects are 35% and 27%, respectively. the average proportion of gene expression variance explained by individually significant snps in the snp-wise regression analysis in this study was 21%. the average proportion of gene expression variance explained by the most significant haplotype partitions at fcgr2b, lmo and pcna was 26%. thus, current technical approaches suggest that approximately one-quarter to one-third of gene expression variation is attributable to cis sequence effects. concordance of the multi-gene cis sequence effects literature and this study pant et al., examined eight genes for cis sequence effects in common with our study, the largest overlap in the extant literature. both studies observed significant cis sequence effects at cyp1b fcgr2b and mybl pant et al., observed ai at tyms where we did not, while we observed cis sequence effects at lmo and nbs where pant et al. did not. neither study observed cis sequence effects at jak and myc. in toto, among the genes jointly analyzed in this study and in the literature, and after excluding two genes with discordant literature results, of genes concordantly exhibit, and of genes concordantly do not exhibit, significant cis sequence effects, respectively. concordance with results obtained using other experimental methods, as we have observed here, increases confidence in our results. such evidence may provide the justification to proceed to more focused functional investigation of gene regulation. the utility of different statistical methods used to evaluate cis sequence effects we used three methods to test for the significance of cis sequence effects, after restricting the number of snps to tag and singleton snps at each gene. regression of individual snps identified the largest number of genes exhibiting significant cis sequence effects and is the most commonly used method in the literature, after assessment of ai. mander's single additive method and templeton's tree scanning method identified three genes; one gene, fcgr2b, was identified in common. the latter two methods incorporate correction for multiple tests in their estimates of the significance of association. rather than apply any formal correction for the multiple comparisons at a gene after regression analysis on snps, or after using multiple methods, we compare and contrast the results obtained from using the three methods. differences between results obtained analyzing individual snps and the two methods that apply multiple test correction at the level of the gene suggests that much of the evidence for significant cis sequence effects in this sample of n = lcls is too weak to survive multiple test correction, emphasizing the necessity to apply multiple test corrections to avoid elevated type i error. we also observed two examples of a gene exhibiting significant cis sequence effects with tree scanning, but not with the additive methods. at lmo the minor alleles of four snps define a haplotype partition that exhibits significantly reduced gene expression, and at pcna, the heterozygote diplotype exhibits significantly increased gene expression. these latter findings depend upon the ability to model the effects of haplotypes within and between individuals. we observed dominant effects for haplotypes at fcgr2b and lmo and heterotic effects for haplotypes at pcna, suggesting that searching for cis sequence effects using only additive model-based approaches may result in elevated type ii error rates. biological relevance of cis sequence effects – fcgr2b as an example the focus of this study is to evaluate association between variation in dna sequence and in vitro rna transcription in a group of candidate genes commonly studied in cancer research. we briefly review some of the recent functional genomics literature for the candidate gene fcgr2b and suggest below how a review of relevant genomic data and our cis sequence effects findings at fcgr2b might inform our understanding of this literature, as an example of how our findings might influence future fcgr2b snp association or functional analyses. the snp500cancer program resequenced portions of ivs exon ivs ivs and exon of fcgr2b, yielding n = fcgr2b snps available with a minor allele frequency of ≥5% for analysis of gene expression variation. after selecting one tag and five singleton snps to reduce the number of statistical tests performed with minimal loss of information, significant cis sequence effects were observed at fcgr2b snp rs. the minor allele frequency of rs in our sample of n = was 10%, similar to the minor allele frequencies of fcgr2b promoter and transmembrane snps previously studied, however, it should be noted that rs is a singleton snp, i.e., not strongly associated with the other snps available at fcgr2b. linkage disequilibrium within the fcgr2b genomic region in caucasian samples extends from the 3' end of ivs distally in a punctate fashion, and there is some evidence for a separate 5' region of ld proximal to exon. rs has been genotyped by hinds et al., as afd with a minor allele frequency of 11%, and within their sample of n = european american dnas, this snp exhibits strong ld with one additional snp. however, both of these snps map to both intronic regions within the fcgr2b locus and also within the fcgr3b/fcgr3a intergenic region some kbp proximal, suggesting that high sequence homology may be interfering with accurate map assignment. there are two recent reports of copy number variation in the region that are relevant: cnv of the fcgr3b locus is associated with nephritis in a rat model and in human patients, and analysis of snp genotypes and genomic hybridization with the hapmap sample has identified a kbp region as human copy number locus cnv_id_ containing the fcgr2a, hspa fcgr3a, fcgr2b and fcrlm loci. our data at fcgr2b is concordant with data generated from both in vitro and ex vivo experimental strategies that sequence variation in the promoter and the coding region is associated with gene expression differences. further, our data contributes to the evidence that a minor allele frequency of ~10% characterizes the snps that are associated with fcgr2b gene expression differences. the inconsistent directionality of effect of the minor allele may be due to high sequence homology at the cnv_id_ locus affecting the physical and linkage disequilibrium mapping of the region, or may be due to incomplete linkage disequilibrium between the analyzed snp and a unanalyzed snp that may be causing the effect. individuals wishing to investigate regulation of gene expression at fcgr2b in the future should include approaches necessary to characterize the physical, linkage disequilibrium, transcriptional and copy number architecture of the region. strengths and limitations strengths of this study include: high quality gene expression data from triplicate cell cultures for each lymphoblastoid cell line, with standardization of culture, rna extraction, labeling, amplification and hybridization conditions; the use of sequence-verified snps and the resequencing of nearly all expression array probe sequence regions; the use of multiple methods to evaluate evidence for significant cis sequence effects; and comparison of the results observed in this study to the published cis sequence effects literature at both the gene and snp levels. limitations of this study include: the use of epstein-barr virus-transformed lcls; the modest number of lcls used; the association paradigm; and the absence of genetic assays that evaluate copy number in our sample. limitations of lcls as reagents for the investigation of gene expression regulation include gene expression in a virally transformed surrogate ex vivo tissue, which may influence and potentially eliminate epstein-barr virus infection associated genes from gene expression investigation. systematic comparison of gene expression from lcls and non-surrogate, minimally processed tissues, e.g., peripheral blood lymphocytes, could be an approach towards validation of gene expression findings made in lcls. the modest number of cell lines used in this study limits statistical power, and is less than the number of cell lines used by some researchers, however, the number of genes evaluated was also modest. the association paradigm suffers from well known limitations. some publications testing large number of genes for cis sequence effects do not provide complete lists of genes tested or of genes exhibiting significant cis sequence effects on gene expression, therefore, we could not identify all genes studied in these reports. also, due to the variety of approaches used in the literature, most comparisons are between categorical results of specific assays, i.e., it is generally not possible to compare quantitative data from different studies. the mybl probe sequence regions were not resequenced in the n = caucasian dna samples and thus the positive regression and treescan results at mybl could potentially be a false positive result due to an unidentified snp within the sequence complementary to these probes.  we tested for significant association between gene sequence variation and gene expression variation at n = candidate genes in dna and rna from n = lcls. significant association between cis sequence and gene expression variation was observed in out of genes, and accounted for 26% of gene expression variation in three genes evaluated using an analysis of variance approach. we utilized additive and analysis of variance analytical approaches, and suggest that approaches that permit modeling of allelic effects may identify effects missed by additive models, although larger multi-gene studies would clarify the relative utility of the two approaches. we reviewed the multi-gene cis sequence effects literature and found data on n = of the candidate genes that we evaluated; most of that data is concordant with our results. investigators using current technologies should expect to find cis sequence effects at about one quarter of candidate genes evaluated: these effects will explain about one quarter of gene expression variance. snps associated with gene expression can be preferentially selected for genotyping and analysis in genetic association studies, or nominated for functional genomic investigations to characterize their role in the regulation of gene expression. 
using the literature as a "gold standard" to compare genes with data from both this study and the literature, we observed 80% and 85% concordance for genes exhibiting and not exhibiting significant cis sequence effects in our study, respectively. based on analysis of our results and the extant literature, one in four genes exhibits significant cis sequence effects, and for these genes, about 30% of gene expression variation is accounted for by cis sequence variation. despite diverse experimental approaches, the presence or absence of significant cis sequence effects is largely supported by previously published studies. we investigated the role of sequence variation in cis on gene expression in a group of genes commonly studied in cancer research in lymphoblastoid cell lines. we generated gene expression profiling data at n = candidate genes from n = lymphoblastoid cell lines for this study and used available candidate gene resequencing data at n = candidate genes to identify n = candidate genes with sufficient variance in both datasets for the investigation of cis sequence effects. the joint analysis of sequence and gene expression variation provides insight into the role of linked sequence variation in the regulation of gene expression. we estimated the proportion of genes exhibiting cis sequence effects and the proportion of gene expression variation explained by cis sequence effects using three different analytical approaches, and compared our results to the literature. snps and diplotypes at eight candidate genes exhibited statistically significant association with gene expression. we used two additive models and the haplotype phylogeny scanning approach of templeton to evaluate association between individual snps, all snps at a gene, and diplotypes, with log-transformed gene expression.
 sequence and transcriptional variability within and between individuals are typically studied independently. the joint analysis of sequence and gene expression variation provides insight into the role of linked sequence variation in the regulation of gene expression. we investigated the role of sequence variation in cis on gene expression in a group of genes commonly studied in cancer research in lymphoblastoid cell lines. we estimated the proportion of genes exhibiting cis sequence effects and the proportion of gene expression variation explained by cis sequence effects using three different analytical approaches, and compared our results to the literature.  we generated gene expression profiling data at n = candidate genes from n = lymphoblastoid cell lines for this study and used available candidate gene resequencing data at n = candidate genes to identify n = candidate genes with sufficient variance in both datasets for the investigation of cis sequence effects. we used two additive models and the haplotype phylogeny scanning approach of templeton to evaluate association between individual snps, all snps at a gene, and diplotypes, with log-transformed gene expression. snps and diplotypes at eight candidate genes exhibited statistically significant association with gene expression. using the literature as a "gold standard" to compare genes with data from both this study and the literature, we observed 80% and 85% concordance for genes exhibiting and not exhibiting significant cis sequence effects in our study, respectively.  based on analysis of our results and the extant literature, one in four genes exhibits significant cis sequence effects, and for these genes, about 30% of gene expression variation is accounted for by cis sequence variation. despite diverse experimental approaches, the presence or absence of significant cis sequence effects is largely supported by previously published studies. 