Experimental modelling of cardiacpressure overload hypertrophy: Modified technique for precise,reproducible, safe and easy aorticarch banding-debanding in mice

Pressure overload left ventricular hypertrophy is a known precursor of heart failure with ominous prognosis. The development of experimental models that reproduce this phenomenon is instrumental for the advancement in our understanding of its pathophysiology. The gold standard of these models is the controlled constriction of the mid aortic arch in mice according to Rockman's technique (RT). We developed a modified technique that allows individualized and fully controlled constriction of the aorta, improves efficiency and generates a reproducible stenosis that is technically easy to perform and release. An algorithm calculates, based on the echocardiographic arch diameter, the intended perimeter at the constriction, and a suture is prepared with two knots separated accordingly. The aorta is encircled twice with the suture and the loop is closed with a microclip under both knots. We performed controlled aortic constriction with Rockman's and the double loop-clip (DLC) techniques in mice. DLC proved superiority in efficiency (mortality and invalid experiments) and more homogeneity of the results (transcoarctational gradients, LV mass, cardiomyocyte hypertrophy, gene expression) than RT. DLC technique optimizes animal use and generates a consistent and customized aortic constriction with homogeneous LV pressure overload morphofunctional, structural, and molecular features.This work was supported by grants from the Ministerio de Economía y Competitividad [Fondo de Investigaciones Sanitarias (PI15/01224), Red de Investigación Cardiovascular (RD12/0042/0018)]. Co-funded by the Fondo Europeo de Desarrollo Regional (FEDER). Fundació La Marató de TV3 (101/C/2015). JFN is afliated to the research network of the Centro de Investigación Biomédica en Red Cardiovascular (CIBERCV), Instituto de Salud Carlos III, Spain

Next-gen sequencing identifies non-coding variation disrupting miRNA-binding sites in neurological disorders

Understanding the genetic factors underlying neurodevelopmental and neuropsychiatric disorders is a major challenge given their prevalence and potential severity for quality of life. While large-scale genomic screens have made major advances in this area, for many disorders the genetic underpinnings are complex and poorly understood. To date the field has focused predominantly on protein coding variation, but given the importance of tightly controlled gene expression for normal brain development and disorder, variation that affects non-coding regulatory regions of the genome is likely to play an important role in these phenotypes. Herein we show the importance of 3 prime untranslated region (3'UTR) non-coding regulatory variants across neurodevelopmental and neuropsychiatric disorders. We devised a pipeline for identifying and functionally validating putatively pathogenic variants from next generation sequencing (NGS) data. We applied this pipeline to a cohort of children with severe specific language impairment (SLI) and identified a functional, SLI-associated variant affecting gene regulation in cells and post-mortem human brain. This variant and the affected gene (ARHGEF39) represent new putative risk factors for SLI. Furthermore, we identified 3'UTR regulatory variants across autism, schizophrenia and bipolar disorder NGS cohorts demonstrating their impact on neurodevelopmental and neuropsychiatric disorders. Our findings show the importance of investigating non-coding regulatory variants when determining risk factors contributing to neurodevelopmental and neuropsychiatric disorders. In the future, integration of such regulatory variation with protein coding changes will be essential for uncovering the genetic causes of complex neurological disorders and the fundamental mechanisms underlying health and disease

CD40L association with protection from severe malaria

CD40 ligand (CD40L), a glycoprotein involved in B cell proliferation, antigen presenting cell activation, and Ig class switching, is important in the immune response to infection. Rare coding mutations in CD40L can lead to life-threatening immunodeficiency but the potential for common variants to alter disease susceptibility remains to be explored. To identify polymorphisms in CD40L, we sequenced 2.3 kb of the 5' flanking region and the first exon of the gene in DNA samples from 36 Gambian females and one chimpanzee. Diversity was lower than the average reported for other areas of the X chromosome, and only two polymorphisms were identified. The polymorphisms were genotyped in DNA samples from 957 Gambian individuals, cases and controls from a study of severe malaria. A significant reduction in risk for severe malaria (OR = 0.52, P = 0.002) was associated with males hemizygous for the CD40L-726C. Analysis by transmission disequilibrium test of 371 cases, for whom DNA from both parents was also available, confirmed the result was not due to stratification (P = 0.04). A similar but non-significant trend was found in females. This preliminary association of a common variant in CD40L with a malaria resistance phenotype encourages further genetic characterization of the role of CD40L in infectious disease