EyeG2P: an automated variant filtering approach improves efficiency of diagnostic genomic testing for inherited ophthalmic disorders.

BACKGROUND: Genomic variant prioritisation is one of the most significant bottlenecks to mainstream genomic testing in healthcare. Tools to improve precision while ensuring high recall are critical to successful mainstream clinical genomic testing, in particular for whole genome sequencing where millions of variants must be considered for each patient. METHODS: We developed EyeG2P, a publicly available database and web application using the Ensembl Variant Effect Predictor. EyeG2P is tailored for efficient variant prioritisation for individuals with inherited ophthalmic conditions. We assessed the sensitivity of EyeG2P in 1234 individuals with a broad range of eye conditions who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we assessed the precision of EyeG2P in comparison with routine diagnostic approaches. For 10 additional individuals, we assessed the utility of EyeG2P for whole genome analysis. RESULTS: EyeG2P had 99.5% sensitivity for genomic variants previously identified as clinically relevant through routine diagnostic analysis (n=1234 individuals). Prospectively, EyeG2P enabled a significant increase in precision (35% on average) in comparison with routine testing strategies (p<0.001). We demonstrate that incorporation of EyeG2P into whole genome sequencing analysis strategies can reduce the number of variants for analysis to six variants, on average, while maintaining high diagnostic yield. CONCLUSION: Automated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders.

Evidence in disease and non-disease contexts that nonsense mutations cause altered splicing via motif disruption.

Transcripts containing premature termination codons (PTCs) can be subject to nonsense-associated alternative splicing (NAS). Two models have been evoked to explain this, scanning and splice motif disruption. The latter postulates that exonic cis motifs, such as exonic splice enhancers (ESEs), are disrupted by nonsense mutations. We employ genome-wide transcriptomic and k-mer enrichment methods to scrutinize this model. First, we show that ESEs are prone to disruptive nonsense mutations owing to their purine richness and paucity of TGA, TAA and TAG. The motif model correctly predicts that NAS rates should be low (we estimate 5-30%) and approximately in line with estimates for the rate at which random point mutations disrupt splicing (8-20%). Further, we find that, as expected, NAS-associated PTCs are predictable from nucleotide-based machine learning approaches to predict splice disruption and, at least for pathogenic variants, are enriched in ESEs. Finally, we find that both in and out of frame mutations to TAA, TGA or TAG are associated with exon skipping. While a higher relative frequency of such skip-inducing mutations in-frame than out of frame lends some credence to the scanning model, these results reinforce the importance of considering splice motif modulation to understand the etiology of PTC-associated disease.

A Depletion of Stop Codons in lincRNA is Owing to Transfer of Selective Constraint from Coding Sequences.

Although the constraints on a gene's sequence are often assumed to reflect the functioning of that gene, here we propose transfer selection, a constraint operating on one class of genes transferred to another, mediated by shared binding factors. We show that such transfer can explain an otherwise paradoxical depletion of stop codons in long intergenic noncoding RNAs (lincRNAs). Serine/arginine-rich proteins direct the splicing machinery by binding exonic splice enhancers (ESEs) in immature mRNA. As coding exons cannot contain stop codons in one reading frame, stop codons should be rare within ESEs. We confirm that the stop codon density (SCD) in ESE motifs is low, even accounting for nucleotide biases. Given that serine/arginine-rich proteins binding ESEs also facilitate lincRNA splicing, a low SCD could transfer to lincRNAs. As predicted, multiexon lincRNA exons are depleted in stop codons, a result not explained by open reading frame (ORF) contamination. Consistent with transfer selection, stop codon depletion in lincRNAs is most acute in exonic regions with the highest ESE density, disappears when ESEs are masked, is consistent with stop codon usage skews in ESEs, and is diminished in both single-exon lincRNAs and introns. Owing to low SCD, the maximum lengths of pseudo-ORFs frequently exceed null expectations. This has implications for ORF annotation and the evolution of de novo protein-coding genes from lincRNAs. We conclude that not all constraints operating on genes need be explained by the functioning of the gene but may instead be transferred owing to shared binding factors.

Adenine Enrichment at the Fourth CDS Residue in Bacterial Genes Is Consistent with Error Proofing for +1 Frameshifts.

Beyond selection for optimal protein functioning, coding sequences (CDSs) are under selection at the RNA and DNA levels. Here, we identify a possible signature of "dual-coding," namely extensive adenine (A) enrichment at bacterial CDS fourth sites. In 99.07% of studied bacterial genomes, fourth site A use is greater than expected given genomic A-starting codon use. Arguing for nucleotide level selection, A-starting serine and arginine second codons are heavily utilized when compared with their non-A starting synonyms. Several models have the ability to explain some of this trend. In part, A-enrichment likely reduces 5' mRNA stability, promoting translation initiation. However T/U, which may also reduce stability, is avoided. Further, +1 frameshifts on the initiating ATG encode a stop codon (TGA) provided A is the fourth residue, acting either as a frameshift "catch and destroy" or a frameshift stop and adjust mechanism and hence implicated in translation initiation. Consistent with both, genomes lacking TGA stop codons exhibit weaker fourth site A-enrichment. Sequences lacking a Shine-Dalgarno sequence and those without upstream leader genes, that may be more error prone during initiation, have greater utilization of A, again suggesting a role in initiation. The frameshift correction model is consistent with the notion that many genomic features are error-mitigation factors and provides the first evidence for site-specific out of frame stop codon selection. We conjecture that the NTG universal start codon may have evolved as a consequence of TGA being a stop codon and the ability of NTGA to rapidly terminate or adjust a ribosome.

A RCT for assessment of active human-centred learning finds teacher-centric non-human teaching of evolution optimal.

Current educational discourse holds that effective pedagogy requires engagement through active student participation with subject matter relating to them. The lack of testing of lessons in series is recognized as a potential weakness in the evidence base, not least because standard parallel designs cannot capture serial interaction effects (cf. drug interactions). However, logistic issues make large-scale replicated in situ assessment of serial designs challenging. The recent introduction of evolution into the UK primary school curriculum presents a rare opportunity to overcome this. We implemented a randomised control 2 × 2 design with four inexpensive schemes of work, comparable to drug interaction trials. This involved an initial test phase (N = 1152) with replication (N = 1505), delivered by teachers, after training, in their classrooms with quantitative before-after-retention testing. Utilising the "genetics-first" approach, the schemes comprised four lessons taught in the same order. Lessons 1 (variation) and 3 (deep-time) were invariant. Lesson 2 (selection) was either student-centred or teacher-centred, with subject organism constant, while lesson 4 (homology) was either human-centred or not, with learning mode constant. All four schemes were effective in replicate, even for lower ability students. Unexpectedly, the teacher-focused/non-human centred scheme was the most successful in both test and replicate, in part owing to a replicable interaction effect but also because it enabled engagement. These results highlight the importance of testing lessons in sequence and indicate that there are many routes to effective engagement with no "one-size fits all" solution in education.

Refining the Ambush Hypothesis: Evidence That GC- and AT-Rich Bacteria Employ Different Frameshift Defence Strategies.

Stop codons are frequently selected for beyond their regular termination function for error control. The "ambush hypothesis" proposes out-of-frame stop codons (OSCs) terminating frameshifted translations are selected for. Although early indirect evidence was partially supportive, recent evidence suggests OSC frequencies are not exceptional when considering underlying nucleotide content. However, prior null tests fail to control amino acid/codon usages or possible local mutational biases. We therefore return to the issue using bacterial genomes, considering several tests defining and testing against a null. We employ simulation approaches preserving amino acid order but shuffling synonymous codons or preserving codons while shuffling amino acid order. Additionally, we compare codon usage in amino acid pairs, where one codon can but the next, otherwise identical codon, cannot encode an OSC. OSC frequencies exceed expectations typically in AT-rich genomes, the +1 frame and for TGA/TAA but not TAG. With this complex evidence, simply rejecting or accepting the ambush hypothesis is not warranted. We propose a refined post hoc model, whereby AT-rich genomes have more accidental frameshifts, handled by RF2-RF3 complexes (associated with TGA/TAA) and are mostly +1 (or -2) slips. Supporting this, excesses positively correlate with in silico predicted frameshift probabilities. Thus, we propose a more viable framework, whereby genomes broadly adopt one of the two strategies to combat frameshifts: preventing frameshifting (GC-rich) or permitting frameshifts but minimizing impacts when most are caught early (AT-rich). Our refined framework holds promise yet some features, such as the bias of out-of-frame sense codons, remain unexplained.

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.