Liver eQTLs
Livers from 312 F2 animals (156 female, 156 male) were profiled using oligonucleotide microarrays manufactured by Agilent Technologies (Palo Alto, California, United States), which included probes for 23,574 mouse transcripts. Individual transcript intensities were corrected for experimental variation and normalized and are reported as the mean log10 ratio (mlratio) of an individual experiment relative to a pool composed of 150 mice randomly selected from the F2 population [21]. Each measurement was fitted to an error model and assigned a significance measurement (type I error). A heat map of the 2,320 transcripts most differentially expressed (p < 0.05 in 10% or more of animals) relative to the pool is depicted in Figure 3. This selection of genes was not biased on a priori known differential expression between the sexes, linkage, or correlation with a clinical phenotype. This is noteworthy because hierarchical clustering of these transcripts against the 312 F2 mice shows an almost perfect clustering into male and female subgroups, emphasizing striking effects of sex on liver gene expression levels and suggesting that sex is controlling more variance in these transcripts' expression than any other parameter.
Figure 3  Heat Map of Liver Gene Expression
Over 2,300 of the most differentially expressed genes in liver hierarchically clustered by animals (x-axis) against transcript levels (y-axis). Expression is reported as mlratio of individual experiment against a common pool. Red is over- and green underrepresented relative to pool. The expression values of the 23,574 transcripts were treated as quantitative traits and fitted to the same linear regression models used to compute LOD scores for clinical traits (eQTLs). The FDR at each threshold was determined by permuting the data 100 times and taking the mean number of QTLs detected over all of the permuted datasets at a given threshold, and dividing this count by the number of QTLs detected at the same threshold in the observed data. At the threshold for significant linkage (p < 5 × 10−5, genome-wide p < 0.05, based on a single trait), the FDR was estimated at 3.4% for the standard QTL model not accounting for any sex terms, 3.1% for the QTL model accounting for additive sex effects, and 3.2% for the QTL model accounting for additive sex effects and allowing for the sex interaction terms to enter the model. A list of all detected suggestive (p < 1 × 10−3) and significant eQTLs (p < 5 × 10−5) detected in the BXH.ApoE−/− intercross is provided in Table S1.
Characteristics of the eQTLs at different significance levels are summarized in Table 3 and shown graphically in Figure 4. We detected 6,676 eQTLs representing 4,998 transcripts at the 5 × 10−5 significant level. Of these, 2,118 eQTLs were located within 20 Mb (roughly 10 cM) of the corresponding gene, likely representing eQTLs regulated by cis-acting variation within the gene itself. Of the 6,676 significant eQTLs, 1,166 (17%) demonstrated a sex bias and were subsequently significantly improved with the addition of the sex-additive and sex-dominant terms.
Table 3  Characteristics of Liver eQTLs
Figure 4  Properties of All Liver eQTLs
(A) Distribution of all significant liver eQTLs across the genome in 2-cM bins. A total of 6,676 significant eQTLs were realized, representing 4,998 liver transcripts. Hotspots of nonrandom eQTL colocalization are clearly evident.
(B) Distribution of eQTLs with significant sex-specific effects. A total of 1,166 eQTLs representing 1,044 transcripts show an eQTL hotspot on Chromosome 5.
(C) Properties of eQTLs at increasing significance levels. As the threshold for significant linkage increases (p-value decreases, or LOD score increases), the proportion of cis-eQTLs (black) increases. The fraction of all eQTLs with sex effects (red) and cis-eQTLs with sex effects (blue) remains relatively constant at increasing thresholds. The dashed line indicates the genome-wide significance threshold (p < 5 × 10−5; genome-wide p < 0.05).
(D) Properties of sex-specific eQTLs at increasing significance levels. For eQTLs with significant sex effects, as with all eQTLs, the proportion of cis-eQTLs (black) increases and trans (blue) decreases as the threshold for significance increases. At the genome-wide threshold for significance (dashed line), over 70% of eQTLs with significant sex effects are trans. The distribution of all 6,676 significant eQTLs (p < 5 × 10−5) across the genome in 2 cM bins is shown in Figure 4A. Evidence for eQTL hotspots is clear on Chromosomes 1, 2, 4, 5, 6, and 7 where significant fractions of the 6,676 eQTLs colocalize within 2 cM regions on each chromosome. Approximately 67% of eQTLs at this threshold are trans, and these eQTL hotspots consist primarily of trans-acting effects on transcriptional variation. Distribution of the 1,166 eQTLs with significant sex effects, of which 852 (73%) are trans, showed enrichment on Chromosome 5 at approximately 49 cM (Figure 4B) as assessed using the Fisher exact test (p = 8.7 × 10−25 after Bonferroni correction). At this locus there were 250 eQTLs, 140 of which exhibited genotype–sex interactions.
At increasing thresholds for linkage, a higher fraction of detected eQTLs were cis-acting (Figure 4C). The increased proportion of cis-eQTLs with increasing LOD score thresholds has been reported before [5,15] and confirms what is likely to be our increased power to detect first-order cis-acting variations affecting transcription. The proportion of eQTLs with significant sex effects remained relatively constant at all thresholds (Figure 4C). Furthermore, the majority of these sex-specific eQTLs (73%) are acting in trans on a given gene's expression (Figure 4D), and similar proportions of sex-specific eQTLs (26%) are cis compared to the proportion of all liver cis-eQTLs (32%). These data demonstrate the profound effects of sex on the genetic regulation of gene expression.