Optimal alpha reduces error rates in gene expression studies: a meta-analysis approach.

BACKGROUND: Transcriptomic approaches (microarray and RNA-seq) have been a tremendous advance for molecular science in all disciplines, but they have made interpretation of hypothesis testing more difficult because of the large number of comparisons that are done within an experiment. The result has been a proliferation of techniques aimed at solving the multiple comparisons problem, techniques that have focused primarily on minimizing Type I error with little or no concern about concomitant increases in Type II errors. We have previously proposed a novel approach for setting statistical thresholds with applications for high throughput omics-data, optimal α, which minimizes the probability of making either error (i.e. Type I or II) and eliminates the need for post-hoc adjustments. RESULTS: A meta-analysis of 242 microarray studies extracted from the peer-reviewed literature found that current practices for setting statistical thresholds led to very high Type II error rates. Further, we demonstrate that applying the optimal α approach results in error rates as low or lower than error rates obtained when using (i) no post-hoc adjustment, (ii) a Bonferroni adjustment and (iii) a false discovery rate (FDR) adjustment which is widely used in transcriptome studies. CONCLUSIONS: We conclude that optimal α can reduce error rates associated with transcripts in both microarray and RNA-seq experiments, but point out that improved statistical techniques alone cannot solve the problems associated with high throughput datasets - these approaches need to be coupled with improved experimental design that considers larger sample sizes and/or greater study replication.

Evolutionary implications of pericentromeric gene expression in humans.

Human pericentromeric sequences are enriched for recent sequence duplications. The continual creation and shuffling of these duplications can create novel intron-exon structures and it has been suggested that these regions have a function as gene nurseries. However, these sequences are also rich in satellite repeats which can repress transcription, and analyses of chromosomes 10 and 21 have suggested that they are transcript poor. Here, we investigate the relationship between pericentromeric duplication and transcription by analyzing the in silico transcriptional profiles within the proximal 1.5 Mb of genomic sequence on all human chromosome arms in relation to duplication status. We identify an approximately 5x excess of transcripts specific to cancer and/or testis in pericentromeric duplications compared to surrounding single copy sequence, with the expression of >50% of all transcripts in duplications being restricted to these tissues. We also identify an approximately 5x excess of transcripts in duplications which contain large quantities of interspersed repeats. These results indicate that the transcriptional profiles of duplicated and single copy sequences within pericentromeric DNA are distinct, suggesting that pericentromeric instability is unlikely to represent a common route for gene creation but may have a disproportionate effect upon genes whose function is restricted to the germ line.

Comparative physical mapping reveals features of microsynteny between Glycine max, Medicago truncatula, and Arabidopsis thaliana.

To gain insight into genomic relationships between soybean (Glycine max) and Medicago truncatula, eight groups of bacterial artificial chromosome (BAC) contigs, together spanning 2.60 million base pairs (Mb) in G. max and 1.56 Mb in M. truncatula, were compared through high-resolution physical mapping combined with sequence and hybridization analysis of low-copy BAC ends. Cross-hybridization among G. max and M. truncatula contigs uncovered microsynteny in six of the contig groups and extensive microsynteny in three. Between G. max homoeologous (within genome duplicate) contigs, 85% of coding and 75% of noncoding sequences were conserved at the level of cross-hybridization. By contrast, only 29% of sequences were conserved between G. max and M. truncatula, and some kilobase-scale rearrangements were also observed. Detailed restriction maps were constructed for 11 contigs from the three highly microsyntenic groups, and these maps suggested that sequence order was highly conserved between G. max duplicates and generally conserved between G. max and M. truncatula. One instance of homoeologous BAC contigs in M. truncatula was also observed and examined in detail. A sequence similarity search against the Arabidopsis thaliana genome sequence identified up to three microsyntenic regions in A. thaliana for each of two of the legume BAC contig groups. Together, these results confirm previous predictions of one recent genome-wide duplication in G. max and suggest that M. truncatula also experienced ancient large-scale genome duplications.

Estimates of conserved microsynteny among the genomes of Glycine max, Medicago truncatula and Arabidopsis thaliana.

A growing body of research indicates that microsynteny is common among dicot genomes. However, most studies focus on just one or a few genomic regions, so the extent of microsynteny across entire genomes remains poorly characterized. To estimate the level of microsynteny between Medicago truncatula (Mt) and Glycine max (soybean), and also among homoeologous segments of soybean, we used a hybridization strategy involving bacterial artificial chromosome (BAC) contigs. A Mt BAC library consisting of 30,720 clones was screened with a total of 187 soybean BAC subclones and restriction fragment length polymorphism (RFLP) probes. These probes came from 50 soybean contig groups, defined as one or more related BAC contigs anchored by the same low-copy probe. In addition, 92 whole soybean BAC clones were hybridized to filters of HindIII-digested Mt BAC DNA to identify additional cases of cross-hybridization after removal of those soybean BACs found to be repetitive in Mt. Microsynteny was inferred when at least two low-copy probes from a single soybean contig hybridized to the same Mt BAC or when a soybean BAC clone hybridized to three or more low-copy fragments from a single Mt BAC. Of the 50 soybean contig groups examined, 54% showed microsynteny to Mt. The degree of conservation among 37 groups of soybean contigs was also investigated. The results indicated substantial conservation among soybean contigs in the same group, with 86.5% of the groups showing at least some level of microsynteny. One contig group was examined in detail by a combination of physical mapping and comparative sequencing of homoeologous segments. A TBLASTX similarity search was performed between 1,085 soybean sequences on the 50 BAC contig groups and the entire Arabidopsis genome. Based on a criterion of sequence homologues <100 kb apart, each with an expected value of < or =1e-07, seven of the 50 soybean contig groups (14%) exhibited microsynteny with Arabidopsis.

Soybean genomic survey: BAC-end sequences near RFLP and SSR markers.

We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.

A prospective study of childhood emotional and behavioural problems in Port Pirie, South Australia.

OBJECTIVE: To describe the extent to which emotional and behavioural problems experienced by 5-year-old children living in or around Port Pirie, South Australia, persisted when the children were aged 11-12 years. METHOD: Childhood emotional and behavioural problems were identified at the age of 5 years using Child Behaviour Checklists completed by mothers. When the children were aged 11-12 years, problems were identified using checklists completed by mothers, children and teachers. RESULTS: Attention problems, aggressive behaviour and anxious/depressed problems were the most persistent problems over this period of the children's lives. In general, the strongest relationship over time occurred when reports were obtained from mothers on each occasion. A weaker relationship existed between earlier mother-reported problems and later teacher-reported problems, while the relationship between mother-reported problems and later self-reported problems occupied an intermediate position. CONCLUSION: The course of problems among children in Port Pirie appeared similar to that previously reported for children in Holland and North America. To better understand the aetiology of psychiatric disorders and to plan for effective interventions, more information is needed about the natural course of childhood emotional and behavioural disorders in Australia.

Lifetime exposure to environmental lead and children's intelligence at 11-13 years: the Port Pirie cohort study.

OBJECTIVE: To examine the association between environmental exposure to lead and children's intelligence at age 11-13 years, and to assess the implications of exposure in the first seven years of life for later childhood development. DESIGN: Prospective cohort study. SUBJECTS: 375 children born in or around the lead smelting town of Port Pirie, Australia, between 1979 and 1982. MAIN OUTCOME MEASURE: Children's intelligence quotient (IQ) measured at 11-13 years of age. RESULTS: IQ was inversely associated with both antenatal and postnatal blood lead concentrations. Verbal, performance, and full scale IQ were inversely related to blood lead concentration with no apparent threshold. Multivariate analyses indicated that after adjustment for a wide range of confounders, the postnatal blood lead concentrations (particularly within the age range 15 months to 7 years) exhibited inverse associations with IQ. Strong associations with IQ were observed for lifetime average blood lead concentrations at various ages. The expected mean full scale IQ declined by 3.0 points (95% confidence interval 0.07 to 5.93) for an increase in lifetime average blood lead concentration from 0.48 to 0.96 mumol/l (10 to 20 micrograms/dl). CONCLUSION: Exposure to environmental lead during the first seven years of life is associated with cognitive deficits that seem to persist into later childhood.

The inhibitory effects of danazol, danazol metabolites, gestrinone, and testosterone on the growth of human endometrial cells in vitro.

Danazol and gestrinone are effective drugs in the treatment of endometriosis. Their mechanism of action remains uncertain, but may be related to their androgenic activity. The authors examined the effect of danazol on human endometrial cells cultured in vitro, its two major metabolites, ethisterone and 2 hydroxymethyl ethisterone, gestrinone, and testosterone (T) at 1X and 10X expected plasma concentrations. Danazol and T suppressed growth by 20.8 and 25.0% (P less than 0.01), respectively, at the lower dose, and by 26.9 and 35.5% (P less than 0.01), respectively, at the 10-fold higher dose. No significant suppression of growth occurred with gestrinone, ethisterone, or 2 hydroxymethyl ethisterone. The results provide further evidence that danazol and T (but not gestrinone) may act by a direct effect on endometrial tissue.

Dosage-related effects of danazol on sex hormone binding globulin and free and total androgen levels.

Danazol is known to cause marked suppression of sex hormone binding globulin (SHBG) levels in plasma and to increase the proportion of plasma testosterone unbound to protein but the effect on the concentration of total and free testosterone is unclear. Twenty-five patients with endometriosis were treated daily for 6 months with doses of danazol ranging from 50 to 600 mg. The fall in SHBG and rise in percent free testosterone was dose-related during the early part of treatment. Suppression of total testosterone and 5 alpha-dihydrotestosterone levels occurred and was probably due to increases in metabolic clearance rates. The observed fall in androstenedione levels was related to the incidence of menstrual abnormality, suggesting that this might be due to reduced ovarian activity. The concentration of free testosterone increased by a factor of two in the first week but subsequently returned to levels of between 25 and 50% above pretreatment levels. This pattern of changes may be due to the rise in metabolic clearance rates being dependent on induction of enzymes of androgen metabolism.

A comparison of the effects of danazol and gestrinone on testosterone binding to sex hormone binding globulin in vitro and in vivo.

Danazol and gestrinone are both effective agents in the treatment of endometriosis. Their mechanism of action is unknown but may be related to their androgenic activity, which is at least partly dependent on increases in the proportion of testosterone which circulates unbound to plasma protein. We have quantified these increases in patients on treatment, and by experimentation in vitro have demonstrated the relative importance of the reduction of sex hormone binding globulin (SHBG) binding capacity and competition with testosterone for SHBG binding sites by the drugs and some of their metabolites. The mean SHBG binding capacity in patients treated with danazol (400 mg/d, n = 7) and gestrinone (5 mg/week, n = 7) fell from 66.9 and 56.4 nmol/l to 36.1 and 28.1 nmol/l, after 1 week's treatment and to 11.1 and 7.1 nmol/l after 4 weeks respectively. Despite the similarity between the falls in SHBG binding capacity there was a significantly greater increase in % free testosterone in plasma samples from patients treated with danazol than in those from patients treated with gestrinone at 1 week. Experiments in vitro suggest that this was largely due to ethisterone (a major metabolite of danazol) competing with testosterone for SHBG binding sites. After 4 weeks on treatment there was a similar, near maximal reduction in SHBG binding of testosterone in both treatment groups. At the low levels of SHBG binding capacity reached by this time the extra effect of any competition for binding sites was much reduced.