A weighted FDR procedure under discrete and heterogeneous null distributions.

Multiple testing (MT) with false discovery rate (FDR) control has been widely conducted in the "discrete paradigm" where p-values have discrete and heterogeneous null distributions. However, in this scenario existing FDR procedures often lose some power and may yield unreliable inference, and for this scenario there does not seem to be an FDR procedure that partitions hypotheses into groups, employs data-adaptive weights and is nonasymptotically conservative. We propose a weighted p-value-based FDR procedure, "weighted FDR (wFDR) procedure" for short, for MT in the discrete paradigm that efficiently adapts to both heterogeneity and discreteness of p-value distributions. We theoretically justify the nonasymptotic conservativeness of the wFDR procedure under independence, and show via simulation studies that, for MT based on p-values of binomial test or Fisher's exact test, it is more powerful than six other procedures. The wFDR procedure is applied to two examples based on discrete data, a drug safety study, and a differential methylation study, where it makes more discoveries than two existing methods.

Environmental Regulation of Heterosis in the Allopolyploid Arabidopsis suecica.

Allopolyploids are organisms possessing more than two complete sets of chromosomes from two or more species and are frequently more vigorous than their progenitors. To address the question why allopolyploids display hybrid vigor, we compared the natural allopolyploid Arabidopsis suecica to its progenitor species Arabidopsis thaliana and Arabidopsis arenosa. We measured chlorophyll content, CO2 assimilation, and carbohydrate production under varying light conditions and found that the allopolyploid assimilates more CO2 per unit chlorophyll than either of the two progenitor species in high intensity light. The increased carbon assimilation corresponds with greater starch accumulation, but only in strong light, suggesting that the strength of hybrid vigor is dependent on environmental conditions. In weaker light A. suecica tends to produce as much primary metabolites as the better progenitor. We found that gene expression of LIMIT DEXTRINASE1, a debranching enzyme that cleaves branch points within starch molecules, is at the same level in the allopolyploid as in the maternal progenitor A. thaliana and significantly more expressed than in the paternal progenitor A. arenosa. However, expression differences of ß-amylases and GLUCAN-WATER DIKINASE1 were not statistically significantly elevated in the allopolyploid over progenitor expression levels. In contrast to allopolyploids, autopolyploid A. thaliana showed the same photosynthetic rate as diploids, indicating that polyploidization alone is likely not the reason for enhanced vigor in the allopolyploid. Taken together, our data suggest that the magnitude of heterosis in A. suecica is environmentally regulated, arises from more efficient photosynthesis, and, under specific conditions, leads to greater starch accumulation than in its progenitor species.

Next-generation transcriptome sequencing of the premenopausal breast epithelium using specimens from a normal human breast tissue bank.

INTRODUCTION: Our efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. In order to provide the specimens that will facilitate such an understanding, The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (KTB) was established. The KTB is, to our knowledge, the only biorepository in the world prospectively established to collect normal, healthy breast tissue from volunteer donors. As a first initiative toward a molecular understanding of the biology and developmental genetics of the normal mammary gland, the effect of the menstrual cycle and hormonal contraceptives on DNA expression in the normal breast epithelium was examined. METHODS: Using normal breast tissue from 20 premenopausal donors to KTB, the changes in the mRNA of the normal breast epithelium as a function of phase of the menstrual cycle and hormonal contraception were assayed using next-generation whole transcriptome sequencing (RNA-Seq). RESULTS: In total, 255 genes representing 1.4% of all genes were deemed to have statistically significant differential expression between the two phases of the menstrual cycle. The overwhelming majority (221; 87%) of the genes have higher expression during the luteal phase. These data provide important insights into the processes occurring during each phase of the menstrual cycle. There was only a single gene significantly differentially expressed when comparing the epithelium of women using hormonal contraception to those in the luteal phase. CONCLUSIONS: We have taken advantage of a unique research resource, the KTB, to complete the first-ever next-generation transcriptome sequencing of the epithelial compartment of 20 normal human breast specimens. This work has produced a comprehensive catalog of the differences in the expression of protein-coding genes as a function of the phase of the menstrual cycle. These data constitute the beginning of a reference data set of the normal mammary gland, which can be consulted for comparison with data developed from malignant specimens, or to mine the effects of the hormonal flux that occurs during the menstrual cycle.

Non-trisomic homeobox gene expression during craniofacial development in the Ts65Dn mouse model of Down syndrome.

Trisomy 21 in humans causes cognitive impairment, craniofacial dysmorphology, and heart defects collectively referred to as Down syndrome. Yet, the pathophysiology of these phenotypes is not well understood. Craniofacial alterations may lead to complications in breathing, eating, and communication. Ts65Dn mice exhibit craniofacial alterations that model Down syndrome including a small mandible. We show that Ts65Dn embryos at 13.5 days gestation (E13.5) have a smaller mandibular precursor but a normal sized tongue as compared to euploid embryos, suggesting a relative instead of actual macroglossia originates during development. Neurological tissues were also altered in E13.5 trisomic embryos. Our array analysis found 155 differentially expressed non-trisomic genes in the trisomic E13.5 mandible, including 20 genes containing a homeobox DNA binding domain. Additionally, Sox9, important in skeletal formation and cell proliferation, was upregulated in Ts65Dn mandible precursors. Our results suggest trisomy causes altered expression of non-trisomic genes in development leading to structural changes associated with DS. Identification of genetic pathways disrupted by trisomy is an important step in proposing rational therapies at relevant time points to ameliorate craniofacial abnormalities in DS and other congenital disorders.

The Collaborative Cross, a community resource for the genetic analysis of complex traits.

The goal of the Complex Trait Consortium is to promote the development of resources that can be used to understand, treat and ultimately prevent pervasive human diseases. Existing and proposed mouse resources that are optimized to study the actions of isolated genetic loci on a fixed background are less effective for studying intact polygenic networks and interactions among genes, environments, pathogens and other factors. The Collaborative Cross will provide a common reference panel specifically designed for the integrative analysis of complex systems and will change the way we approach human health and disease.

Epigenomic consequences of immortalized plant cell suspension culture.

Plant cells grown in culture exhibit genetic and epigenetic instability. Using a combination of chromatin immunoprecipitation and DNA methylation profiling on tiling microarrays, we have mapped the location and abundance of histone and DNA modifications in a continuously proliferating, dedifferentiated cell suspension culture of Arabidopsis. We have found that euchromatin becomes hypermethylated in culture and that a small percentage of the hypermethylated genes become associated with heterochromatic marks. In contrast, the heterochromatin undergoes dramatic and very precise DNA hypomethylation with transcriptional activation of specific transposable elements (TEs) in culture. High throughput sequencing of small interfering RNA (siRNA) revealed that TEs activated in culture have increased levels of 21-nucleotide (nt) siRNA, sometimes at the expense of the 24-nt siRNA class. In contrast, TEs that remain silent, which match the predominant 24-nt siRNA class, do not change significantly in their siRNA profiles. These results implicate RNA interference and chromatin modification in epigenetic restructuring of the genome following the activation of TEs in immortalized cell culture.

Ghost QTL and hotspots in experimental crosses: novel approach for modeling polygenic effects.

Ghost quantitative trait loci (QTL) are the false discoveries in QTL mapping, that arise due to the "accumulation" of the polygenic effects, uniformly distributed over the genome. The locations on the chromosome that are strongly correlated with the total of the polygenic effects depend on a specific sample correlation structure determined by the genotypes at all loci. The problem is particularly severe when the same genotypes are used to study multiple QTL, e.g. using recombinant inbred lines or studying the expression QTL. In this case, the ghost QTL phenomenon can lead to false hotspots, where multiple QTL show apparent linkage to the same locus. We illustrate the problem using the classic backcross design and suggest that it can be solved by the application of the extended mixed effect model, where the random effects are allowed to have a nonzero mean. We provide formulas for estimating the thresholds for the corresponding t-test statistics and use them in the stepwise selection strategy, which allows for a simultaneous detection of several QTL. Extensive simulation studies illustrate that our approach eliminates ghost QTL/false hotspots, while preserving a high power of true QTL detection.

Gene expression analysis at the intersection of ploidy and hybridity in maize.

Heterosis and polyploidy are two important aspects of plant evolution. To examine these issues, we conducted a global gene expression study of a maize ploidy series as well as a set of tetraploid inbred and hybrid lines. This gene expression analysis complements an earlier phenotypic study of these same materials. We find that ploidy change affects a large fraction of the genome, albeit at low levels; gene expression changes rarely exceed 2-fold and are typically not statistically significant. The most common gene expression profile we detected is greater than linear increase from monoploid to diploid, and reductions from diploid to triploid and from triploid to tetraploid, a trend that mirrors plant stature. When examining heterosis in tetraploid maize lines, we found a large fraction of the genome impacted but the majority of changes were not statistically significant at 2-fold or less. Non-additive expression was common in the hybrids, and the extent of non-additivity increased both in number and magnitude from duplex to quadruplex hybrids. Overall, we find that gene expression trends mirror observations from the phenotypic studies; however, obvious mechanistic connections remain unknown.

Epigenetic natural variation in Arabidopsis thaliana.

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F(2) families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.

Presynaptic calcium channel localization and calcium-dependent synaptic vesicle exocytosis regulated by the Fuseless protein.

A systematic forward genetic Drosophila screen for electroretinogram mutants lacking synaptic transients identified the fuseless (fusl) gene, which encodes a predicted eight-pass transmembrane protein in the presynaptic membrane. Null fusl mutants display >75% reduction in evoked synaptic transmission but, conversely, an approximately threefold increase in the frequency and amplitude of spontaneous synaptic vesicle fusion events. These neurotransmission defects are rescued by a wild-type fusl transgene targeted only to the presynaptic cell, demonstrating a strictly presynaptic requirement for Fusl function. Defects in FM dye turnover at the synapse show a severely impaired exo-endo synaptic vesicle cycling pool. Consistently, ultrastructural analyses reveal accumulated vesicles arrested in clustered and docked pools at presynaptic active zones. In the absence of Fusl, calcium-dependent neurotransmitter release is dramatically compromised and there is little enhancement of synaptic efficacy with elevated external Ca(2+) concentrations. These defects are causally linked with severe loss of the Cacophony voltage-gated Ca(2+) channels, which fail to localize normally at presynaptic active zone domains in the absence of Fusl. These data indicate that Fusl regulates assembly of the presynaptic active zone Ca(2+) channel domains required for efficient coupling of the Ca(2+) influx and synaptic vesicle exocytosis during neurotransmission.

Naive application of permutation testing leads to inflated type I error rates.

Failure to account for family structure within populations or in complex mating designs via uninformed applications of permutation testing will lead to inflated type I error rates. Careful consideration of the design factors is essential since some situations allow several valid permutation strategies, and the choice that maximizes statistical power will not always be intuitive.

Cholesterol Sulfotransferase SULT2B1b Modulates Sensitivity to Death Receptor Ligand TNFα in Castration-Resistant Prostate Cancer.

Cholesterol sulfotransferase, SULT2B1b, has been demonstrated to modulate both androgen receptor activity and cell growth properties. However, the mechanism(s) by which SULT2B1b alters these properties within prostate cancer cells has not been described. Furthermore, specific advantages of SULT2B1b expression in prostate cancer cells are not understood. In these studies, single-cell mRNA sequencing was conducted to compare the transcriptomes of SULT2B1b knockdown (KD) versus Control KD LNCaP cells. Over 2,000 differentially expressed genes were identified along with alterations in numerous canonical pathways, including the death receptor signaling pathway. The studies herein demonstrate that SULT2B1b KD increases TNFα expression in prostate cancer cells and results in NF-κB activation in a TNF-dependent manner. More importantly, SULT2B1b KD significantly enhances TNF-mediated apoptosis in both TNF-sensitive LNCaP cells and TNF-resistant C4-2 cells. Overexpression of SULT2B1b in LNCaP cells also decreases sensitivity to TNF-mediated cell death, suggesting that SULT2B1b modulates pathways dictating the TNF sensitivity capacity of prostate cancer cells. Probing human prostate cancer patient datasets further supports this work by providing evidence that SULT2B1b expression is inversely correlated with TNF-related genes, including TNF, CD40LG, FADD, and NFKB1. Together, these data provide evidence that SULT2B1b expression in prostate cancer cells enhances resistance to TNF and may provide a growth advantage. In addition, targeting SULT2B1b may induce an enhanced therapeutic response to TNF treatment in advanced prostate cancer. IMPLICATIONS: These data suggest that SULT2B1b expression enhances resistance to TNF and may promote prostate cancer.

Global eQTL mapping reveals the complex genetic architecture of transcript-level variation in Arabidopsis.

The genetic architecture of transcript-level variation is largely unknown. The genetic determinants of transcript-level variation were characterized in a recombinant inbred line (RIL) population (n = 211) of Arabidopsis thaliana using whole-genome microarray analysis and expression quantitative trait loci (eQTL) mapping of transcript levels as expression traits (e-traits). Genetic control of transcription was highly complex: one-third of the quantitatively controlled transcripts/e-traits were regulated by cis-eQTL, and many trans-eQTL mapped to hotspots that regulated hundreds to thousands of e-traits. Several thousand eQTL of large phenotypic effect were detected, but almost all (93%) of the 36,871 eQTL were associated with small phenotypic effects (R(2) < 0.3). Many transcripts/e-traits were controlled by multiple eQTL with opposite allelic effects and exhibited higher heritability in the RILs than their parents, suggesting nonadditive genetic variation. To our knowledge, this is the first large-scale global eQTL study in a relatively large plant mapping population. It reveals that the genetic control of transcript level is highly variable and multifaceted and that this complexity may be a general characteristic of eukaryotes.

Extending the modified bayesian information criterion (mBIC) to dense markers and multiple interval mapping.

SUMMARY: The modified version of Bayesian Information Criterion (mBIC) is a relatively simple model selection procedure that can be used when locating multiple interacting quantitative trait loci (QTL). Our earlier work demonstrated the statistical properties of mBIC for situations where the average genetic map interval is at least 5 cM. In this work mBIC is adapted to genome searches based on a dense map and, more importantly, to the situation where consecutive QTL and interactions are located by multiple interval mapping. Easy to use formulas for the extended mBIC are given. A simulation study, as well as the analysis of real data, confirm the good properties of the extended mBIC.

QTL mapping in rice.

In the past 10 years, interest in applying the tools of molecular genetics to the problem of increasing world rice production has resulted in the generation of two highly saturated, molecular linkage maps of rice, and the localization of numerous genes and quantitative trait loci (QTLs). Primary studies have identified QTLs associated with disease resistance, abiotic stress tolerance and yield potential of rice in a range of ecosystems. The ability to identify, manipulate and potentially clone individual genes involved in quantitatively inherited characters, combined with the demonstrated conservation of numerous linkage blocks among members of the grass family, emphasizes the contribution of map-based genetic analyses both to applied and to basic crop research.

Understanding mechanisms of novel gene expression in polyploids.

Polyploidy has long been recognized as a prominent force shaping the evolution of eukaryotes, especially flowering plants. New phenotypes often arise with polyploid formation and can contribute to the success of polyploids in nature or their selection for use in agriculture. Although the causes of novel variation in polyploids are not well understood, they could involve changes in gene expression through increased variation in dosage-regulated gene expression, altered regulatory interactions, and rapid genetic and epigenetic changes. New research approaches are being used to study these mechanisms and the results should provide a more complete understanding of polyploidy.

eae36, a locus on mouse chromosome 4, controls susceptibility to experimental allergic encephalomyelitis in older mice and mice immunized in the winter.

Genetic factors are believed to contribute to multiple sclerosis (MS) susceptibility; however, strong evidence implicating intrinsic and environmental factors in the etiopathogenesis of MS also exists. Susceptibility to experimental allergic encephalomyelitis (EAE), the principal animal model of MS, is also influenced by nongenetic factors, including age and season at immunization. This suggests that age- and season-by-gene interactions exist and that different susceptibility loci may influence disease as a function of the two parameters. In this study, linkage analysis based on genome exclusion mapping was carried out using age and season at immunization restricted cohorts of (B10.S x SJL/J) F2 intercross mice in an effort to identify such linkages. Significant linkage of EAE to eae4 and eae5 was detected with 6- to 12-week-old and summer cohorts. In contrast, significant linkage of EAE to eae4 and eae5 was not detected with the >12-week-old and winter/spring populations. Rather, significant linkage to D4Mit203 at 128.50 Mb on chromosome 4 was detected with animals that were >12 weeks old at the time of immunization or were immunized in the winter. This previously unidentified locus has been designated eae36. These results support the existence of age- and season-by-gene-specific interactions in the genetic control of susceptibility to autoimmune inflammatory disease of the central nervous system and suggest that late-onset MS may be immunogenetically distinct.

Genomewide nonadditive gene regulation in Arabidopsis allotetraploids.

Polyploidy has occurred throughout the evolutionary history of all eukaryotes and is extremely common in plants. Reunification of the evolutionarily divergent genomes in allopolyploids creates regulatory incompatibilities that must be reconciled. Here we report genomewide gene expression analysis of Arabidopsis synthetic allotetraploids, using spotted 70-mer oligo-gene microarrays. We detected >15% transcriptome divergence between the progenitors, and 2105 and 1818 genes were highly expressed in Arabidopsis thaliana and A. arenosa, respectively. Approximately 5.2% (1362) and 5.6% (1469) genes displayed expression divergence from the midparent value (MPV) in two independently derived synthetic allotetraploids, suggesting nonadditive gene regulation following interspecific hybridization. Remarkably, the majority of nonadditively expressed genes in the allotetraploids also display expression changes between the parents, indicating that transcriptome divergence is reconciled during allopolyploid formation. Moreover, >65% of the nonadditively expressed genes in the allotetraploids are repressed, and >94% of the repressed genes in the allotetraploids match the genes that are expressed at higher levels in A. thaliana than in A. arenosa, consistent with the silencing of A. thaliana rRNA genes subjected to nucleolar dominance and with overall suppression of the A. thaliana phenotype in the synthetic allotetraploids and natural A. suecica. The nonadditive gene regulation is involved in various biological pathways, and the changes in gene expression are developmentally regulated. In contrast to the small effects of genome doubling on gene regulation in autotetraploids, the combination of two divergent genomes in allotetraploids by interspecific hybridization induces genomewide nonadditive gene regulation, providing a molecular basis for de novo variation and allopolyploid evolution.

Genomic survey of gene expression diversity in Arabidopsis thaliana.

Differential gene expression controls variation in numerous plant traits, such as flowering time and plant/pest interactions, but little is known about the genomic distribution of the determinants of transcript levels and their associated variation. Affymetrix ATH1 GeneChip microarrays representing 22,810 genes were used to survey the transcriptome of seven Arabidopsis thaliana accessions in the presence and absence of exogenously applied salicylic acid (SA). These accessions encompassed approximately 80% of the moderate- to high-frequency nucleotide polymorphisms in Arabidopsis. A factorial design, consisting of three biological replicates per accession for the two treatments at three time points (4, 28, and 52 hr post-treatment), and a total of 126 microarrays were used. Between any pair of Arabidopsis accessions, we detected on average 2234 genes (ranging from 1428 to 3334) that were significantly differentially expressed under the conditions of this experiment, using a split-plot analysis of variance. Upward of 6433 genes were differentially expressed between at least one pair of accessions. These results suggest that analysis of additional genetic, developmental, and environmental conditions may show that a significant fraction of the Arabidopsis genome is differentially expressed. Examination of sequence diversity demonstrated a significant positive association with diversity in gene expression.

Novel Resampling Improves Statistical Power for Multiple-Trait QTL Mapping.

Multiple-trait analysis typically employs models that associate a quantitative trait locus (QTL) with all of the traits. As a result, statistical power for QTL detection may not be optimal if the QTL contributes to the phenotypic variation in only a small proportion of the traits. Excluding QTL effects that contribute little to the test statistic can improve statistical power. In this article, we show that an optimal power can be achieved when the number of QTL effects is best estimated, and that a stringent criterion for QTL effect selection may improve power when the number of QTL effects is small but can reduce power otherwise. We investigate strategies for excluding trivial QTL effects, and propose a method that improves statistical power when the number of QTL effects is relatively small, and fairly maintains the power when the number of QTL effects is large. The proposed method first uses resampling techniques to determine the number of nontrivial QTL effects, and then selects QTL effects by the backward elimination procedure for significance test. We also propose a method for testing QTL-trait associations that are desired for biological interpretation in applications. We validate our methods using simulations and Arabidopsis thaliana transcript data.