The Genom of Homo sapiens.pdf

112 COTSAPAS ET AL.Figure 5. The detection of cis or trans origins of difference inmRNA levels of a gene. The location of the gene is indicated bya dashed line: If the amounts of mRNA are controlled in cis, themRNA levels (arrows labeled cis) are concordant with the originof the cognate gene. Conversely, if the levels are controlledin trans (arrows labeled trans), then they are concordant withthe origin of the transcription factor (TF) and not with the cognategene itself.els of mRNA (TRANS in figure) will correlate with theparental origin of TF, NOT the gene: In this case, TF is B-derived in all five lines. In practice, the level of mRNA ismeasured with microarrays.Our experimental approach was to create pools of totalRNA derived from brain tissue from three animals ofeach strain. Each pool was reverse-transcribed, fluorescentlylabeled, and competitively hybridized with a referencecDNA sample from a pool of three C57BL/6J animals.All animals were age- and sex-matched. The arrays,produced by the Ramaciotti Centre for Gene FunctionAnalysis (UNSW, Sydney, Australia), were spotted withthe 15K NIA cDNA clone set.Statistical Analysis of cis VariationWe have analyzed these array hybridizations using astatistical approach implemented with custom-writtensoftware. In outline, we perform lowess print tip normalizationof background-subtracted fluorescence intensityratios (RI/B) to define relative hybridization for eachcDNA (Yang et al. 2002).Using the computed SDP for the relevant gene, M valuesfrom each RI are assigned to B or D as appropriate,and a t statistic is calculated to assess the difference betweenthe two groups. To assess significance, we permuteall possible combinations of M values for the number ofBs and Ds in the SDP, and calculate t statistics for each.By placing the experimental t statistic onto this distribution,we can derive a percentile (P) value: essentially, ameasure of probability that the fit between the data andthe SDP is derived by chance. The importance of this approachis that it requires no a priori assumptions of differencelevels between samples and can be used to assessdifferences between the parental strains or within the RIlines. Using a variety of data based on observation, wehave modeled this process and conclude that we have~40% power (with confidence interval, C.I., of 99.8%) todetect 1.5-fold variation of signal with a variance of 1,which increases to >68% (with C.I. of 99.8%) as the folddifference is increased to >2. These observations agreewith anecdotal evidence as to the resolution power of microarrays(Claverie 1999).Trans variation is identified by a similar process, but incalculating t-statistics we input all SDPs known to occurwithin the RI lines (there are 807 of these), rather than usingthe single cognate gene SDP, and searching for a significantmatch. In other words, we ask whether anygene’s strain distribution pattern correlates with the fluctuationof transcript level from the RI lines, with the expectationthat a trans effector will do so. This approachgenerates substantial problems of multiple sampling andcomputation time, and we necessarily have to preselectthe cDNAs to reduce the confounding multiple testing,using a number of criteria. We are currently limiting thisanalysis to cDNAs we believe to be differentially expressedbetween the parental strains themselves; concurrently,we are developing statistical approaches to analyzethe complete, non-selected data set.Experimental Analysis of the Originof Quantitative VariationWe have used three approaches to determine the numberof genes contained within the NIA 15K set that are differentiallyexpressed between the parental C57BL/6J andDBA/2J strains. Eight replicates of B/D microarray datawere analyzed using the three different methods to identifycandidate differentially expressed genes in the parentallines: by a simple heuristic (8 replicates > 2-fold [2/8min]), 36 genes (0.3%), by a t-statistic with a 0.1% cutoff,241 genes (1.7%), and by B-statistic (Lonnstedt and Speed2002), 13 genes (0.09%) are differentially expressed betweenC57BL/6J and DBA/2J. There is overlap betweenthese analyses yielding 279 unique candidate genes. Eachof these genes was examined in the RI series and expressionpatterns were assessed against the known SDP of thegene. Of the 192 genes that could be analyzed, only 5(2.6%) had cis determinants with a 99.8% confidence.Strikingly, 8 additional genes, not differentially expressedbetween the parental strains, could be shown inthe RI strains to exhibit cis variation at 99.8% confidence,which suggests to us that genetic background is critical indetermining the mRNA levels.Trans analysis: The 279 genes were analyzed using thet-statistic approach by matching the individual RI strains’M values against all 808 SDPs contained within the RIset. Of 210,000 matches, 412 were significant at the99.8% level, and these contained 56% of the differentiallyexpressed genes.Our interpretation of these preliminary data is that ofthe genes exhibiting differential expression, ~60% aresusceptible to monogenic cis or trans influences, whereas~40% are due to oligogenic, polygenic, or other influencesthat cannot readily be mapped in these analyses.We also detect, but cannot presently quantitate, significantepistatic influences in those genes that are differentiallyexpressed in the RI lines but not the parental strains.Identifying a RegulonA regulon is a group of genes that are coordinately expressedbecause they share a common control mecha-