Lumbar Spine Fusion and Symptoms of Leg Length Discrepancy After Hip Arthroplasty.

BACKGROUND: Hip length discrepancy (HLD) is common after total hip arthroplasty (THA); however, the effect of spinal fusion on perceived leg length discrepancy (LLD) symptoms after THA has not been examined. This study tested the hypothesis that LLD symptoms are increased in patients who underwent lumbar spinal fusion and THA, compared with patients with THA only. METHODS: This retrospective cohort study included 67 patients who underwent lumbar spinal fusion and THA, along with 78 matched control patients who underwent THA only. Hip and spine measurements were taken on postoperative, standing anterior-posterior pelvic, lateral lumbar, and anterior-posterior lumbar spinal radiographs. Perceived LLD symptoms were assessed via telephone survey. RESULTS: Between the spinal fusion and control groups, there was no significant difference in HLD (M = 7.10 mm, SE = 0.70 and M = 5.60 mm, SE = 0.49) (P = .403). The spinal fusion patients reported more frequently noticing a difference in the length of their legs than the control group (P = .046) and reported limping "all the time" compared with the control group (P = .001). Among all patients with an HLD ≤10 mm, those in the spinal fusion group reported limping at a higher frequency than patients in the control group (P = .008). Patients in the spinal fusion group were also more likely to report worsened back pain after THA (P = .011) than the control group. CONCLUSION: Frequencies of a perceived LLD, limping, and worsened back pain after THA were increased in patients with THA and a spinal fusion compared with patients who had THA only, even in a population with HLD traditionally considered to be subclinical. The results indicate that in patients with prior spinal fusion, precautions should be taken to avoid even minor LLD in the setting of THA.

ENCODE whole-genome data in the UCSC Genome Browser.

The Encyclopedia of DNA Elements (ENCODE) project is an international consortium of investigators funded to analyze the human genome with the goal of producing a comprehensive catalog of functional elements. The ENCODE Data Coordination Center at The University of California, Santa Cruz (UCSC) is the primary repository for experimental results generated by ENCODE investigators. These results are captured in the UCSC Genome Bioinformatics database and download server for visualization and data mining via the UCSC Genome Browser and companion tools (Rhead et al. The UCSC Genome Browser Database: update 2010, in this issue). The ENCODE web portal at UCSC (http://encodeproject.org or http://genome.ucsc.edu/ENCODE) provides information about the ENCODE data and convenient links for access.

The UCSC Genome Browser database: update 2010.

The University of California, Santa Cruz (UCSC) Genome Browser website (http://genome.ucsc.edu/) provides a large database of publicly available sequence and annotation data along with an integrated tool set for examining and comparing the genomes of organisms, aligning sequence to genomes, and displaying and sharing users' own annotation data. As of September 2009, genomic sequence and a basic set of annotation 'tracks' are provided for 47 organisms, including 14 mammals, 10 non-mammal vertebrates, 3 invertebrate deuterostomes, 13 insects, 6 worms and a yeast. New data highlights this year include an updated human genome browser, a 44-species multiple sequence alignment track, improved variation and phenotype tracks and 16 new genome-wide ENCODE tracks. New features include drag-and-zoom navigation, a Wiki track for user-added annotations, new custom track formats for large datasets (bigBed and bigWig), a new multiple alignment output tool, links to variation and protein structure tools, in silico PCR utility enhancements, and improved track configuration tools.

Large-scale appearance of ultraconserved elements in tetrapod genomes and slowdown of the molecular clock.

Mammalian genomes contain millions of highly conserved noncoding sequences, many of which are regulatory. The most extreme examples are the 481 ultraconserved elements (UCEs) that are identical over at least 200 bp in human, mouse, and rat and show 96% identity with chicken, which diverged approximately 310 MYA. If the substitution rate in UCEs remained constant, these elements should also be present with a high level of identity in fish (approximately 450 Myr), but this is not the case, suggesting that many appeared in the amniotes or tetrapods or that the molecular clock has slowed down in these lineages, or both. Taking advantage of the availability of multiple genomes, we identified 13,736 UCEs in the human genome that are identical over at least 100 bp in at least 3 of 5 placental mammals, including 2,189 sequences over at least 200 bp, thereby greatly expanding the repertoire of known UCEs, and investigated the evolution of these sequences in opossum, chicken, frog, and fish. We conclude that there was a massive genome-wide acquisition and expansion of UCEs during tetrapod and then amniote evolution, accompanied by a slowdown of the molecular clock, particularly in the amniotes, a process consistent with their functional exaptation in these lineages. The majority of tetrapod-specific UCEs are noncoding and associated with genes involved in regulation of transcription and development. In contrast, fish genomes contain relatively few UCEs, the majority of which are common to all bony vertebrates. These elements are different from other conserved noncoding elements and appear to be important regulatory innovations that became fixed following the emergence of vertebrates from the sea to the land.

Maintenance of transposon-free regions throughout vertebrate evolution.

BACKGROUND: We recently reported the existence of large numbers of regions up to 80 kb long that lack transposon insertions in the human, mouse and opossum genomes. These regions are significantly associated with loci involved in developmental and transcriptional regulation. RESULTS: Here we report that transposon-free regions (TFRs) are prominent genomic features of amphibian and fish lineages, and that many have been maintained throughout vertebrate evolution, although most transposon-derived sequences have entered these lineages after their divergence. The zebrafish genome contains 470 TFRs over 10 kb and a further 3,951 TFRs over 5 kb, which is comparable to the number identified in mammals. Two thirds of zebrafish TFRs over 10 kb are orthologous to TFRs in at least one mammal, and many have orthologous TFRs in all three mammalian genomes as well as in the genome of Xenopus tropicalis. This indicates that the mechanism responsible for the maintenance of TFRs has been active at these loci for over 450 million years. However, the majority of TFR bases cannot be aligned between distantly related species, demonstrating that TFRs are not the by-product of strong primary sequence conservation. Syntenically conserved TFRs are also more enriched for regulatory genes compared to lineage-specific TFRs. CONCLUSION: We suggest that TFRs contain extended regulatory sequences that contribute to the precise expression of genes central to early vertebrate development, and can be used as predictors of important regulatory regions.

Evidence for control of splicing by alternative RNA secondary structures in Dipteran homothorax pre-mRNA.

In a recent study that identified highly evolutionary conserved sequences in three genomes of Diptera species we described an ultraconserved element found at an internal exon-intron junction of the Drosophila melanogaster homothorax (hth) gene that appeared to be involved in the control of hth pre-mRNA splicing. We also discussed a possible role of RNA secondary structure at this site in the regulation of hth pre-mRNA splicing. In this report we identify a shorter evolutionary conserved intronic element within the hth gene that is located downstream of the first element and has sequence complementarity to it. We demonstrate that intramolecular interactions between these two elements would give rise to alternative RNA secondary structures, which in turn may result in differential control of homothorax pre-mRNA splicing. We also provide additional comparative genomic data from several newly available insect genomes supporting our original conclusion that these conserved elements are important in the post-transcriptional regulation of homothorax gene expression in Diptera.

Genomics Virtual Laboratory: A Practical Bioinformatics Workbench for the Cloud.

BACKGROUND: Analyzing high throughput genomics data is a complex and compute intensive task, generally requiring numerous software tools and large reference data sets, tied together in successive stages of data transformation and visualisation. A computational platform enabling best practice genomics analysis ideally meets a number of requirements, including: a wide range of analysis and visualisation tools, closely linked to large user and reference data sets; workflow platform(s) enabling accessible, reproducible, portable analyses, through a flexible set of interfaces; highly available, scalable computational resources; and flexibility and versatility in the use of these resources to meet demands and expertise of a variety of users. Access to an appropriate computational platform can be a significant barrier to researchers, as establishing such a platform requires a large upfront investment in hardware, experience, and expertise. RESULTS: We designed and implemented the Genomics Virtual Laboratory (GVL) as a middleware layer of machine images, cloud management tools, and online services that enable researchers to build arbitrarily sized compute clusters on demand, pre-populated with fully configured bioinformatics tools, reference datasets and workflow and visualisation options. The platform is flexible in that users can conduct analyses through web-based (Galaxy, RStudio, IPython Notebook) or command-line interfaces, and add/remove compute nodes and data resources as required. Best-practice tutorials and protocols provide a path from introductory training to practice. The GVL is available on the OpenStack-based Australian Research Cloud (http://nectar.org.au) and the Amazon Web Services cloud. The principles, implementation and build process are designed to be cloud-agnostic. CONCLUSIONS: This paper provides a blueprint for the design and implementation of a cloud-based Genomics Virtual Laboratory. We discuss scope, design considerations and technical and logistical constraints, and explore the value added to the research community through the suite of services and resources provided by our implementation.

Comparison of ultra-conserved elements in drosophilids and vertebrates.

Metazoan genomes contain many ultra-conserved elements (UCEs), long sequences identical between distant species. In this study we identified UCEs in drosophilid and vertebrate species with a similar level of phylogenetic divergence measured at protein-coding regions, and demonstrated that both the length and number of UCEs are larger in vertebrates. The proportion of non-exonic UCEs declines in distant drosophilids whilst an opposite trend was observed in vertebrates. We generated a set of 2,126 Sophophora UCEs by merging elements identified in several drosophila species and compared these to the eutherian UCEs identified in placental mammals. In contrast to vertebrates, the Sophophora UCEs are depleted around transcription start sites. Analysis of 52,954 P-element, piggyBac and Minos insertions in the D. melanogaster genome revealed depletion of the P-element and piggyBac insertions in and around the Sophophora UCEs. We examined eleven fly strains with transposon insertions into the intergenic UCEs and identified associated phenotypes in five strains. Four insertions behave as recessive lethals, and in one case we observed a suppression of the marker gene within the transgene, presumably by silenced chromatin around the integration site. To confirm the lethality is caused by integration of transposons we performed a phenotype rescue experiment for two stocks and demonstrated that the excision of the transposons from the intergenic UCEs restores viability. Sequencing of DNA after the transposon excision in one fly strain with the restored viability revealed a 47 bp insertion at the original transposon integration site suggesting that the nature of the mutation is important for the appearance of the phenotype. Our results suggest that the UCEs in flies and vertebrates have both common and distinct features, and demonstrate that a significant proportion of intergenic drosophila UCEs are sensitive to disruption.

Raising the estimate of functional human sequences.

While less than 1.5% of the mammalian genome encodes proteins, it is now evident that the vast majority is transcribed, mainly into non-protein-coding RNAs. This raises the question of what fraction of the genome is functional, i.e., composed of sequences that yield functional products, are required for the expression (regulation or processing) of these products, or are required for chromosome replication and maintenance. Many of the observed noncoding transcripts are differentially expressed, and, while most have not yet been studied, increasing numbers are being shown to be functional and/or trafficked to specific subcellular locations, as well as exhibit subtle evidence of selection. On the other hand, analyses of conservation patterns indicate that only approximately 5% (3%-8%) of the human genome is under purifying selection for functions common to mammals. However, these estimates rely on the assumption that reference sequences (usually ancient transposon-derived sequences) have evolved neutrally, which may not be the case, and if so would lead to an underestimate of the fraction of the genome under evolutionary constraint. These analyses also do not detect functional sequences that are evolving rapidly and/or have acquired lineage-specific functions. Indeed, many regulatory sequences and known functional noncoding RNAs, including many microRNAs, are not conserved over significant evolutionary distances, and recent evidence from the ENCODE project suggests that many functional elements show no detectable level of sequence constraint. Thus, it is likely that much more than 5% of the genome encodes functional information, and although the upper bound is unknown, it may be considerably higher than currently thought.

The relationship between non-protein-coding DNA and eukaryotic complexity.

There are two intriguing paradoxes in molecular biology--the inconsistent relationship between organismal complexity and (1) cellular DNA content and (2) the number of protein-coding genes--referred to as the C-value and G-value paradoxes, respectively. The C-value paradox may be largely explained by varying ploidy. The G-value paradox is more problematic, as the extent of protein coding sequence remains relatively static over a wide range of developmental complexity. We show by analysis of sequenced genomes that the relative amount of non-protein-coding sequence increases consistently with complexity. We also show that the distribution of introns in complex organisms is non-random. Genes composed of large amounts of intronic sequence are significantly overrepresented amongst genes that are highly expressed in the nervous system, and amongst genes downregulated in embryonic stem cells and cancers. We suggest that the informational paradox in complex organisms may be explained by the expansion of cis-acting regulatory elements and genes specifying trans-acting non-protein-coding RNAs.

Orthologous microRNA genes are located in cancer-associated genomic regions in human and mouse.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNAs that regulate differentiation and development in many organisms and play an important role in cancer. METHODOLOGY/PRINCIPAL FINDINGS: Using a public database of mapped retroviral insertion sites from various mouse models of cancer we demonstrate that MLV-derived retroviral inserts are enriched in close proximity to mouse miRNA loci. Clustered inserts from cancer-associated regions (Common Integration Sites, CIS) have a higher association with miRNAs than non-clustered inserts. Ten CIS-associated miRNA loci containing 22 miRNAs are located within 10 kb of known CIS insertions. Only one CIS-associated miRNA locus overlaps a RefSeq protein-coding gene and six loci are located more than 10 kb from any RefSeq gene. CIS-associated miRNAs on average are more conserved in vertebrates than miRNAs associated with non-CIS inserts and their human homologs are also located in regions perturbed in cancer. In addition we show that miRNA genes are enriched around promoter and/or terminator regions of RefSeq genes in both mouse and human. CONCLUSIONS/SIGNIFICANCE: We provide a list of ten miRNA loci potentially involved in the development of blood cancer or brain tumors. There is independent experimental support from other studies for the involvement of miRNAs from at least three CIS-associated miRNA loci in cancer development.

Transposon-free regions in mammalian genomes.

Despite the presence of over 3 million transposons separated on average by approximately 500 bp, the human and mouse genomes each contain almost 1000 transposon-free regions (TFRs) over 10 kb in length. The majority of human TFRs correlate with orthologous TFRs in the mouse, despite the fact that most transposons are lineage specific. Many human TFRs also overlap with orthologous TFRs in the marsupial opossum, indicating that these regions have remained refractory to transposon insertion for long evolutionary periods. Over 90% of the bases covered by TFRs are noncoding, much of which is not highly conserved. Most TFRs are not associated with unusual nucleotide composition, but are significantly associated with genes encoding developmental regulators, suggesting that they represent extended regions of regulatory information that are largely unable to tolerate insertions, a conclusion difficult to reconcile with current conceptions of gene regulation.

Ultraconserved elements in insect genomes: a highly conserved intronic sequence implicated in the control of homothorax mRNA splicing.

Recently, we identified a large number of ultraconserved (uc) sequences in noncoding regions of human, mouse, and rat genomes that appear to be essential for vertebrate and amniote ontogeny. Here, we used similar methods to identify ultraconserved genomic regions between the insect species Drosophila melanogaster and Drosophila pseudoobscura, as well as the more distantly related Anopheles gambiae. As with vertebrates, ultraconserved sequences in insects appear to occur primarily in intergenic and intronic sequences, and at intron-exon junctions. The sequences are significantly associated with genes encoding developmental regulators and transcription factors, but are less frequent and are smaller in size than in vertebrates. The longest identical, nongapped orthologous match between the three genomes was found within the homothorax (hth) gene. This sequence spans an internal exon-intron junction, with the majority located within the intron, and is predicted to form a highly stable stem-loop RNA structure. Real-time quantitative PCR analysis of different hth splice isoforms and Northern blotting showed that the conserved element is associated with a high incidence of intron retention in hth pre-mRNA, suggesting that the conserved intronic element is critically important in the post-transcriptional regulation of hth expression in Diptera.

Ultraconserved elements in the human genome.

There are 481 segments longer than 200 base pairs (bp) that are absolutely conserved (100% identity with no insertions or deletions) between orthologous regions of the human, rat, and mouse genomes. Nearly all of these segments are also conserved in the chicken and dog genomes, with an average of 95 and 99% identity, respectively. Many are also significantly conserved in fish. These ultraconserved elements of the human genome are most often located either overlapping exons in genes involved in RNA processing or in introns or nearby genes involved in the regulation of transcription and development. Along with more than 5000 sequences of over 100 bp that are absolutely conserved among the three sequenced mammals, these represent a class of genetic elements whose functions and evolutionary origins are yet to be determined, but which are more highly conserved between these species than are proteins and appear to be essential for the ontogeny of mammals and other vertebrates.