Fine-Scale Variation and Genetic Determinants of Alternative Splicing across Individuals

Recently, thanks to the increasing throughput of new technologies, we have begun to explore the full extent of alternative pre–mRNA splicing (AS) in the human transcriptome. This is unveiling a vast layer of complexity in isoform-level expression differences between individuals. We used previously published splicing sensitive microarray data from lymphoblastoid cell lines to conduct an in-depth analysis on splicing efficiency of known and predicted exons. By combining publicly available AS annotation with a novel algorithm designed to search for AS, we show that many real AS events can be detected within the usually unexploited, speculative majority of the array and at significance levels much below standard multiple-testing thresholds, demonstrating that the extent of cis-regulated differential splicing between individuals is potentially far greater than previously reported. Specifically, many genes show subtle but significant genetically controlled differences in splice-site usage. PCR validation shows that 42 out of 58 (72%) candidate gene regions undergo detectable AS, amounting to the largest scale validation of isoform eQTLs to date. Targeted sequencing revealed a likely causative SNP in most validated cases. In all 17 incidences where a SNP affected a splice-site region, in silico splice-site strength modeling correctly predicted the direction of the micro-array and PCR results. In 13 other cases, we identified likely causative SNPs disrupting predicted splicing enhancers. Using Fst and REHH analysis, we uncovered significant evidence that 2 putative causative SNPs have undergone recent positive selection. We verified the effect of five SNPs using in vivo minigene assays. This study shows that splicing differences between individuals, including quantitative differences in isoform ratios, are frequent in human populations and that causative SNPs can be identified using in silico predictions. Several cases affected disease-relevant genes and it is likely some of these differences are involved in phenotypic diversity and susceptibility to complex diseases

Prognostic significance of nucleolar assessment in invasive breast cancer

Aims: Nucleolar morphometric features have a potential role in the assessment of aggressiveness of many cancers. However, the role of nucleoli in invasive breast cancer (IBC) is still unclear. This study aimed to investigate the optimal scoring method of nucleoli in IBC and their prognostic significance, and refine the grading of BC by incorporating the nucleolar score.Methods and results: Digital images acquired from hematoxylin and eosin (H&E) stained sections from a large IBC cohort were divided into training (n=400) and validation (n=1200) sets were used in this study. Four different assessment methods including 1) modified Helpap’s method, and counting prominent nucleoli (size ≥2.5μm) in 2) 10 field views (10 FVs), 3) 5 FVs and 4) 1 FV were evaluated in the training set to identify the optimal method associated with the best performance and significant prognostic value. The optimal method was applied to the validation set and to an external validation set the Cancer Genome Atlas (TCGA) data (n=743). Scoring prominent nucleoli in 5 FVs, showed the highest inter-observer concordance rate (intraclass correlation coefficient = 0.8) and significant association with breast cancer specific survival (BCSS) (

White matter integrity in Tanzanian children with sickle cell anemia: A diffusion tensor imaging study

Background and Purpose: widespread reductions in white matter integrity are associated with cognitive dysfunction in sickle cell anemia. Silent cerebral infarction (SCI), vasculopathy (VSC), and low hemoglobin concentration (Hb) are implicated; we aimed to determine independent contributions to microstructural white matter injury and whether white matter integrity differs across arterial territories. Methods: sixty two children with sickle cell anemia aged 6 to 19 years were prospectively studied at Muhimbili National Hospital, Tanzania. SCI± and VSC± were identified on magnetic resonance imaging (MRI)/magnetic resonance angiography (MRA) scans by 2 neuroradiologists. Tract-based spatial statistics tested for voxel-wise differences in diffusion tensor imaging metrics (ie, fractional anisotropy, mean diffusivity, radial diffusivity, and axial diffusivity) between SCI± and VSC± groups, with correlations between diffusion tensor imaging metrics and Hb. In tract-based spatial statistics analyses, potentially mediating factors (ie, age, sex, as well as Hb, SCI, and/or vasculopathy) were covariates. Differences in mean diffusion tensor imaging metrics across regions of interest in arterial territories were explored. Results: compared with SCI-patients (n=45), SCI+ patients (n=17) exhibited increased radial diffusivity in multiple regions; negative relationships were observed between mean diffusivity, axial diffusivity, and Hb (P&lt;0.005). Compared with VSC-patients (n=49), mild (n=6) or moderate (n=7) VSC+ patients exhibited reduced fractional anisotropy in widespread regions (P&lt;0.05) including the anterior longitudinal fasciculi, corpus callosum, internal capsule, corona radiata, and corticospinal tracts. Overall, the posterior cerebral arterial territory had higher mean mean diffusivity and mean radial diffusivity than the anterior and middle cerebral arterial territories, although no patient had vasculopathy in this area. There was an interaction between territory and vasculopathy. Conclusions: SCI, vasculopathy, and Hb are independent risk factors, and thus treatment targets, for diffuse white matter injury in patients with sickle cell anemia. Exacerbation of hemodynamic stress may play a role.</p