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Biomedical advances have led to a relaxation of natural selection in the human population in developed countries. In the absence of strong purifying selection, spontaneous and frequently deleterious mutations tend to accumulate in the human genome and gradually increase the genetic load; that is, the frequency of potentially lethal genes in the gene pool. Gradual increase in incidence of many hereditary disorders suggests deleterious impact of the genetic load on human well being. Many complex diseases such as type 1 and 2 diabetes, autism, bipolar disorder, allergies, Alzheimer disease, and some cancers show significantly higher concordance in monozygotic (MZ) twins than in dizygotic twins (DZ) or parent-child pairs, suggesting their etiology is strongly influenced by genetics. Preventing these diseases based on genetic data alone is frequently impossible. We hypothesize that the incidence of complex diseases could be significantly reduced in the future through a strategy based on time-separated twinning. This strategy involves the collection and fertilization of human oocytes followed by several rounds of artificial twinning. If preimplantation genetic screening (PGS) reports no aneuploidy or known Mendelian disorders, one of the MZ siblings would be implanted and the remaining embryos cryoconserved. Once the health of the adult MZ sibling(s) is established, subsequent parenthood with the cryoconserved twins could substantially lower the incidence of hereditary disorders with Mendelian or complex etiology. The proposed method of artificial twinning has the potential to alleviate suffering and reduce the negative social impact induced by dysgenic effects associated with known and unknown genetic factors. Time-separated twinning has the capacity to prevent further accumulation of the genetic load and to provide source of isogenic embryonic stem cells for future regenerative therapies., Supporting web site http://prevmed.big.ac.cn contains more information. Poster on the project is available at http://f1000.com/posters/browse/summary/1092806

Motivation: Microsatellites are among the most useful genetic markers in population biology. High-throughput sequencing of microsatellite-enriched libraries dramatically expedites the traditional process of screening recombinant libraries for microsatellite markers. However, sorting through millions of reads to distill high-quality polymorphic markers requires special algorithms tailored to tolerate sequencing errors in locus reconstruction, distinguish paralogous loci, rarify raw reads originating from the same amplicon and sort out various artificial fragments resulting from recombination or concatenation of auxiliary adapters. Existing programs warrant improvement. Results: We describe a microsatellite prediction framework named HighSSR for microsatellite genotyping based on high-throughput sequencing. We demonstrate the utility of HighSSR in comparison to Roche gsAssembler on two Roche 454 GS FLX runs. The majority of the HighSSR-assembled loci were reliably mapped against model organism reference genomes. HighSSR demultiplexes pooled libraries, assesses locus polymorphism and implements Primer3 for the design of PCR primers flanking polymorphic microsatellite loci. As sequencing costs drop and permit the analysis of all project samples on next-generation platforms, this framework can also be used for direct simple sequence repeats genotyping. Availability: http://code.google.com/p/highssr/ Contact: alexander@big.ac.cn Supplementary Information: Supplementary data are available at Bioinformatics online.

Genetic nonself recognition systems such as vegetative incompatibility operate in many filamentous fungi to regulate hyphal fusion between genetically dissimilar individuals and to restrict the spread of virulence-attenuating mycoviruses that have potential for biological control of pathogenic fungi. We report here the use of a comparative genomics approach to identify seven candidate polymorphic genes associated with four vegetative incompatibility (vic) loci of the chestnut blight fungus Cryphonectria parasitica. Disruption of candidate alleles in one of two strains that were heteroallelic at vic2, vic6, or vic7 resulted in enhanced virus transmission, but did not prevent barrage formation associated with mycelial incompatibility. Detailed characterization of the vic6 locus revealed the involvement of nonallelic interactions between two tightly linked genes in barrage formation, heterokaryon formation, and asymmetric, gene-specific influences on virus transmission. The combined results establish molecular identities of genes associated with four C. parasitica vic loci and provide insights into how these recognition factors interact to trigger incompatibility and restrict virus transmission.

Contains fulltext : 116540.pdf (Publisher’s version ) (Open Access) BACKGROUND: Angiotensin-converting enzyme (ACE) (EC 4.15.1) metabolizes many biologically active peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels are associated with different cardiovascular and respiratory diseases. METHODS AND RESULTS: Two Belgian families with a 8-16-fold increase in blood ACE level were incidentally identified. A novel heterozygous splice site mutation of intron 25 - IVS25+1G>A (c.3691+1G>A) - cosegregating with elevated plasma ACE was identified in both pedigrees. Messenger RNA analysis revealed that the mutation led to the retention of intron 25 and Premature Termination Codon generation. Subjects harboring the mutation were mostly normotensive, had no left ventricular hypertrophy or cardiovascular disease. The levels of renin-angiotensin-aldosterone system components in the mutated cases and wild-type controls were similar, both at baseline and after 50 mg captopril. Compared with non-affected members, quantification of ACE surface expression and shedding using flow cytometry assay of dendritic cells derived from peripheral blood monocytes of affected members, demonstrated a 50% decrease and 3-fold increase, respectively. Together with a dramatic increase in circulating ACE levels, these findings argue in favor of deletion of transmembrane anchor, leading to direct secretion of ACE out of cells. CONCLUSIONS: We describe a novel mutation of the ACE gene associated with a major familial elevation of circulating ACE, without evidence of activation of the renin-angiotensin system, target organ damage or cardiovascular complications. These data are consistent with the hypothesis that membrane-bound ACE, rather than circulating ACE, is responsible for Angiotensin II generation and its cardiovascular consequences. 12 p.

GC 5' splice sites (5'ss) are present in ∼1% of human introns, but factors promoting their efficient selection are poorly understood. Here, we describe a case of X-linked agammaglobulinemia resulting from a GC 5'ss activated by a mutation in BTK intron 3. This GC 5'ss was intrinsically weak, yet it was selected in >90% primary transcripts in the presence of a strong and intact natural GT counterpart. We show that efficient selection of this GC 5'ss required a high density of GAA/CAA-containing splicing enhancers in the exonized segment and was promoted by SR proteins 9G8, Tra2β and SC35. The GC 5'ss was efficiently inhibited by splice-switching oligonucleotides targeting either the GC 5'ss itself or the enhancer. Comprehensive analysis of natural GC-AG introns and previously reported pathogenic GC 5'ss showed that their efficient activation was facilitated by higher densities of splicing enhancers and lower densities of silencers than their GT 5'ss equivalents. Removal of the GC-AG introns was promoted to a minor extent by the splice-site strength of adjacent exons and inhibited by flanking Alu repeats, with the first downstream Alus located on average at a longer distance from the GC 5'ss than other transposable elements. These results provide new insights into the splicing code that governs selection of noncanonical splice sites.

Background Polymorphic variants and mutations disrupting canonical splicing isoforms are among the leading causes of human hereditary disorders. While there is a substantial evidence of aberrant splicing causing Mendelian diseases, the implication of such events in multi-genic disorders is yet to be well understood. We have developed a new tool (SpliceScan II) for predicting the effects of genetic variants on splicing and cis-regulatory elements. The novel Bayesian non-canonical 5'GC splice site (SS) sensor used in our tool allows inference on non-canonical exons. Results Our tool performed favorably when compared with the existing methods in the context of genes linked to the Autism Spectrum Disorder (ASD). SpliceScan II was able to predict more aberrant splicing isoforms triggered by the mutations, as documented in DBASS5 and DBASS3 aberrant splicing databases, than other existing methods. Detrimental effects behind some of the polymorphic variations previously associated with Alzheimer's and breast cancer could be explained by changes in predicted splicing patterns. Conclusions We have developed SpliceScan II, an effective and sensitive tool for predicting the detrimental effects of genomic variants on splicing leading to Mendelian and complex hereditary disorders. The method could potentially be used to screen resequenced patient DNA to identify de novo mutations and polymorphic variants that could contribute to a genetic disorder.

Total evidence and the use of large datasets to overcome uncertainty are the state of the art in systematic analysis. This assumes that the only true phylogenetic signal is ancestry and that functional, structural, and other factors will not add an alternative signal. Using gene families, where individual codon positions were sorted into bins based upon average-pairwise dN/dS ratio, we show that standard, common phylogenetic methods that were designed for stochastic, neutral, site-independent processes, generate less robust phylogenetic signal for bins with strong negative or positive selection. This was true for phylogenetic reconstruction with parsimony, distance, and likelihood methods. Further, we present a case for the potential existence of systematic functional or structural signal that competes with ancestral signal. For the example of positive selection, we simulate the evolution of sequences through three dimensional lattice constructs with folding constraint and changing binding functionality and show that total evidence for these lattice genes presents trees with functional signal, but that the neutral synonymous sites in these genes show the true ancestral signal. In this case, sequence convergence is promoted by functional convergence.

Background: The fat body is the main organ of intermediary metabolism in insects and the principal source of hemolymph proteins. As part of our ongoing efforts to understand mosquito fat body physiology and to identify novel targets for insect control, we have conducted a transcriptome analysis of the fat body of Aedes aegypti before and in response to blood feeding. Results: We created two fat body non-normalized EST libraries, one from mosquito fat bodies non-blood fed (NBF) and another from mosquitoes 24 hrs post-blood meal (PBM). 454 pyrosequencing of the non-normalized libraries resulted in 204,578 useable reads from the NBF sample and 323,474 useable reads from the PBM sample. Alignment of reads to the existing reference Ae. aegypti transcript libraries for analysis of differential expression between NBF and PBM samples revealed 116,912 and 115,051 matches, respectively. De novo assembly of the reads from the NBF sample resulted in 15,456 contigs, and assembly of the reads from the PBM sample resulted in 15,010 contigs. Collectively, 123 novel transcripts were identified within these contigs. Prominently expressed transcripts in the NBF fat body library were represented by transcripts encoding ribosomal proteins. Thirty-five point four percent of all reads in the PBM library were represented by transcripts that encode yolk proteins. The most highly expressed were transcripts encoding members of the cathepsin b, vitellogenin, vitellogenic carboxypeptidase, and vitelline membrane protein families. Conclusion: The two fat body transcriptomes were considerably different from each other in terms of transcript expression in terms of abundances of transcripts and genes expressed. They reflect the physiological shift of the pre-feeding fat body from a resting state to vitellogenic gene expression after feeding.

Background Auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from the University of California Santa Cruz multiple genome alignments of human and 22 Tetrapoda organisms to predict candidate enhancers and silencers that have reproducible and statistically significant bias towards annotated exonic boundaries. Results A total of 2,546 Tetrapoda enhancers and silencers were clustered into 15 putative core motifs based on their Markov properties. Most of these elements have been identified previously, but 118 putative silencers and 260 enhancers (~15%) were novel. Examination of previously published experimental data for the presence of predicted elements showed that their mutations in 21/23 (91.3%) cases altered the splicing pattern as expected. Predicted intronic motifs flanking 3' and 5' splice sites had higher evolutionary conservation than other sequences within intronic flanks and the intronic enhancers were markedly differed between 3' and 5' intronic flanks. Conclusion Difference in intronic enhancers supporting 5' and 3' splice sites suggests an independent splicing commitment for neighboring exons. Increased evolutionary conservation for ISEs/ISSs within intronic flanks and effect of modulation of predicted elements on splicing suggest functional significance of found elements in splicing regulation. Most of the elements identified were shown to have direct implications in human splicing and therefore could be useful for building computational splicing models in biomedical research.