Comparative analysis of gene duplications and their impact on expression levels in nematode genomes

Gene duplication is a major mechanism that plays a vital role in different evolutionary innovations, ranging from generating novel traits to phenotypic plasticity. Evolutionary impact of gene duplication and the fate of duplicated genes has been studied in detail. However, little is known about the impact of gene duplication on gene expression with respect to different evolutionary time scales. Here, we study genome-wide patterns of gene duplications in nematodes and assess their effect on expression levels. This study encompasses various macroevolutionary comparisons at different time scales and microevolutionary comparisons within the species Pristionchus pacificus. At the macroevolutionary level, by comparing species separated more than 280 million years ago, we found various lineage-specific expansions in multiple gene families along the Pristionchus lineage. Moreover, we found that duplicated genes are highly enriched among developmentally regulated genes. Interestingly, the results also show evidence for selection on duplication to increases the gene expression levels in a developmental stage-specific manner. To gain insights into the microevolution of gene expression levels after gene duplication, we compared different strains of P.pacificus and found that an additional gene copy does usually not increase gene expression levels in the different strains. Furthermore, we found a strong depletion of duplicated genes in large parts of the P. pacificus genome indicating towards negative selection against gene duplication. This shows that the impact on gene expression levels following gene duplication differs dramatically, where a selection for increased gene dosage dominates macroevolution and negative selection on gene duplication dominates within species level. This led us to wonder what happens at the intermediate time scale. We compared recent duplicates of P. pacificus with their single-copy orthologs in two closely related species and found a pattern similar to the microevolutionary trend. Additionally, comparison of closely related species of the Strongyloides genus and its developmental transcriptome also shows overall strong depletion of duplicated genes, similar to the observation at the microevolutionary level. At the same time, a strong enrichment of duplicated genes was found at a developmental stage associated with the parasitic activity of the nematodes. Similar to the macroevolutionary picture of P. pacificus, we also found selection for higher gene dosage in parasitism-associated gene families of S. papillosus, indicating the adaptive potential of duplicated genes. Even though these studies show widespread selection against both duplication and changes in gene expression, duplications are favoured in some conditions leading to adaptive changes in the organism. Overall this indicates that the regulation of expression levels of duplicated genes was subjected to different selection processes at different time scales, which represent a complex interplay between different evolutionary processes like natural selection, population dynamics, and genetic drift

Ancient gene duplications have shaped developmental stage-specific expression in Pristionchus pacificus

BACKGROUND: The development of multicellular organisms is accompanied by gene expression changes in differentiating cells. Profiling stage-specific expression during development may reveal important insights into gene sets that contributed to the morphological diversity across the animal kingdom. RESULTS: We sequenced RNA-seq libraries throughout a developmental timecourse of the nematode Pristionchus pacificus. The transcriptomes reflect early larval stages, adult worms including late larvae, and growth-arrested dauer larvae and allowed the identification of developmentally regulated gene clusters. Our data reveals similar trends as previous transcriptome profiling of dauer worms and represents the first expression data for early larvae in P. pacificus. Gene expression clusters characterizing early larval stages show most significant enrichments of chaperones, while collagens are most significantly enriched in transcriptomes of late larvae and adult worms. By combining expression data with phylogenetic analysis, we found that developmentally regulated genes are found in paralogous clusters that have arisen through lineage-specific duplications after the split from the Caenorhabditis elegans branch. CONCLUSIONS: We propose that gene duplications of developmentally regulated genes represent a plausible evolutionary mechanism to increase the dosage of stage-specific expression. Consequently, this may contribute to the substantial divergence in expression profiles that has been observed across larger evolutionary time scales

The Impact of Visual Impairment on Functional Vision of Children in Rural South India: The Kariapatti Pediatric Eye Evaluation Project

PURPOSE. To determine the impact of visual impairment on functional vision of children in a rural population of south India. METHODS. A visual function questionnaire (LVP-VFQ) was administered to 1194 children aged 7 to 15 years identified through a systematic random sampling technique from 144 hamlets of Kariapatti in rural south India as part of a larger population-based project. Visual acuity estimations and clinical examinations for morbidity were performed in these 1194 children. A Rasch analysis was performed to validate the use of the instrument in this population. Bootstrap estimates (95% confidence intervals) of the regression coefficients were used to compare visual function scores between children with normal sight and children with uncorrected monocular and binocular visual impairment

Deoxyribonucleic acid methylation profiling of single human blastocysts by methylated CpG-island amplification coupled with CpG-island microarray

Objective To study whether methylated CpG-island (CGI) amplification coupled with microarray (MCAM) can be used to generate DNA (deoxyribonucleic acid) methylation profiles from single human blastocysts. Design A pilot microarray study with methylated CpG-island amplification applied to human blastocyst genomic DNA and hybridized on CpG-island microarrays. Setting University research laboratory. Patient(s) Five cryopreserved sibling 2-pronuclear zygotes that were surplus to requirements for clinical treatment by in vitro fertilization were donated with informed consent from a patient attending Bourn Hall Clinic, Cambridge, United Kingdom. Intervention(s) None. Main Outcome Measure(s) Successful generation of genome-wide DNA methylation profiles at CpG islands from individual human blastocysts, with common genomic regions of DNA methylation identified between embryos. Result(s) Between 472 and 734 CpG islands were methylated in each blastocyst, with 121 CpG islands being commonly methylated in all 5 blastocysts. A further 159 CGIs were commonly methylated in 4 of the 5 tested blastocysts. Methylation was observed at a number of CGIs within imprinted-gene, differentially methylated regions (DMRs), including placental and preimplantation-specific DMRs. Conclusion(s) The MCAM method is capable of providing comprehensive DNA methylation data in individual human blastocysts

Microevolution of Duplications and Deletions and Their Impact on Gene Expression in the Nematode Pristionchus pacificus.

The evolution of diversity across the animal kingdom has been accompanied by tremendous gene loss and gain. While comparative genomics has been fruitful to characterize differences in gene content across highly diverged species, little is known about the microevolution of structural variations that cause these differences in the first place. In order to investigate the genomic impact of structural variations, we made use of genomic and transcriptomic data from the nematode Pristionchus pacificus, which has been established as a satellite model to Caenorhabditis elegans for comparative biology. We exploit the fact that P. pacificus is a highly diverse species for which various genomic data including the draft genome of a sister species P. exspectatus is available. Based on resequencing coverage data for two natural isolates we identified large (> 2 kb) deletions and duplications relative to the reference strain. By restriction to completely syntenic regions between P. pacificus and P. exspectatus, we were able to polarize the comparison and to assess the impact of structural variations on expression levels. We found that while loss of genes correlates with lack of expression, duplication of genes has virtually no effect on gene expression. Further investigating expression of individual copies at sites that segregate between the duplicates, we found in the majority of cases only one of the copies to be expressed. Nevertheless, we still find that certain gene classes are strongly depleted in deletions as well as duplications, suggesting evolutionary constraint acting on synteny. In summary, our results are consistent with a model, where most structural variations are either deleterious or neutral and provide first insights into the microevolution of structural variations in the P. pacificus genome

Depletion of SVs among highly conserved genes.

<p>We classified genes in deleted and duplicated regions (relative to the reference strain PS312) based on presence of protein domains as well as homology relationships with other nematodes including <i>C. elegans</i>. For the different homology classes, we find that predicted one-to-one orthologs with <i>C. elegans</i> are strongly depleted from SVs indicating the action of purifying selection. Conversely, conserved and orphan genes that are members of larger gene families are significantly enriched in SVs suggesting that similar processes that have generated different homology relationships at a cross-species level are operating on a microevolutionary level.</p

Identification of duplications and deletions in the <i>P. pacificus</i> strains.

<p>A) General phylogeny of <i>P. pacificus</i> strains and the sister species <i>P. exspectatus</i> (RS5522). Strains and species that are used in this study are colored in red. The tree was generated by building a neighbor joining tree on Hamming distances based on roughly 450,000 parsimony informative sites (singletons excluded). Excluding singletons leads to a vast underestimation of distances between strains and species but should increase the robustness of the tree topology. All internal nodes showed a perfect bootstrap support of 100/100. B) True and false positive rate for SV calls for various p-value and fold change cutoffs of the program cnv-seq. The evaluation is based on 100 manually classified SV calls per strain per SV type and final cutoff combinations were chosen subjectively. C) The graphs show the fraction of genomic sequence within non-overlapping 100kb windows, predicted to be duplicated (positive values) and deleted (negative values) relative to the reference strain PS312. Contigs were concatenated using the genetic map of <i>P. pacificus</i> to produce chromosome-scale plots [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131136#pone.0131136.ref036" target="_blank">36</a>]. Gray boxes indicate conserved syntenic regions with the sister species <i>P. exspectatus</i>. While duplications exhibit a much more even distribution, we identified two almost megabase sized regions with high fraction of missing sequence on the X chromosome.</p

Gene families enriched in SVs.

<p>We defined gene families based on the presence of protein domains (PFAM) and tested whether genes of a given gene family are enriched in SVs. Panels A and D shows significantly enriched gene families for deletions and duplications relative to the reference strain PS312 as pooled data (unpolarized analysis). The other panels show the enriched gene families after restricting to perfectly collinear regions and interpreting the SVs as derived events (polarized analysis).</p

Depletion of SVs among highly conserved genes.

<p>We classified genes in deleted and duplicated regions (relative to the reference strain PS312) based on presence of protein domains as well as homology relationships with other nematodes including <i>C. elegans</i>. For the different homology classes, we find that predicted one-to-one orthologs with <i>C. elegans</i> are strongly depleted from SVs indicating the action of purifying selection. Conversely, conserved and orphan genes that are members of larger gene families are significantly enriched in SVs suggesting that similar processes that have generated different homology relationships at a cross-species level are operating on a microevolutionary level.</p

Effect of duplications and deletions on gene expression levels.

<p>Distribution of expression categories of duplicated and deleted genes for the reference strain and the strain of interest. Only genes within perfectly collinear regions between the reference genome and the sister species were used for this analysis. While deletions lead to a strong increase in genes without expression, i.e. more than 80% (RS5200) and 65% (RS5410) of genes in predicted deletions show indeed no evidence of expression. In contrast, we do not see the opposite trend for duplicated genes.</p