Reverse genetic screen for loss-of-function mutations uncovers a frame-shifting deletion in the melanophilin gene accountable for a distinctive coat-color in Belgian Blue Cattle.

RILOUK

Mapping QTL influencing gastrointestinal nematode burden in Dutch Holstein-Friesian dairy cattle

BACKGROUND: Parasitic gastroenteritis caused by nematodes is only second to mastitis in terms of health costs to dairy farmers in developed countries. Sustainable control strategies complementing anthelmintics are desired, including selective breeding for enhanced resistance. RESULTS AND CONCLUSION: To quantify and characterize the genetic contribution to variation in resistance to gastro-intestinal parasites, we measured the heritability of faecal egg and larval counts in the Dutch Holstein-Friesian dairy cattle population. The heritability of faecal egg counts ranged from 7 to 21% and was generally higher than for larval counts. We performed a whole genome scan in 12 paternal half-daughter groups for a total of 768 cows, corresponding to the approximately 10% most and least infected daughters within each family (selective genotyping). Two genome-wide significant QTL were identified in an across-family analysis, respectively on chromosomes 9 and 19, coinciding with previous findings in orthologous chromosomal regions in sheep. We identified six more suggestive QTL by within-family analysis. An additional 73 informative SNPs were genotyped on chromosome 19 and the ensuing high density map used in a variance component approach to simultaneously exploit linkage and linkage disequilibrium in an initial inconclusive attempt to refine the QTL map position

Dp71 expression in human glioblastoma

Background: Dp71 is the most abundant dystrophin (DMD) gene product in the nervous system. Mutation in the Dp71 coding region is associated with cognitive disturbances in Duchenne muscular dystrophy (DMD) patients, but the function of dystrophin Dp71 in tumor progression remains to be established. This study investigated Dp71 expression in glioblastoma, the most common and aggressive primary tumor of the central nervous system (CNS). Methods: Dp71 expression was analyzed by immunofluorescence, immunohistochemistry, RT-PCR, and immunoblotting in glioblastoma cell lines and cells isolated from human glioblastoma multiforme (GBM) bioptic specimens. Results: Dp71 isoform was expressed in normal human astrocytes (NHA) cell lines and decreased in glioblastoma cell lines and cells isolated from human glioblastoma multiforme bioptic specimens. Moreover, Dp71 was localized in the nucleus in normal cells, while it was localized into the cytoplasm of glioblastoma cells organized in clusters. We have shown, by double labeling, that Dp71 colocalizes with lamin B in normal astrocytes cells, confirming the roles of Dp71 and lamin B in maintaining nuclear architecture. Finally, we demonstrated that decreased Dp71 protein in cells isolated from human bioptic specimens was inversely correlated with the Ki-67 tumor proliferative index. Conclusion: A decreased Dp71 expression is associated with cancer proliferation and poor prognosis in glioblastoma

Selection in action: dissecting the molecular underpinnings of the increasing muscle mass of Belgian Blue Cattle.

BACKGROUND: Belgian Blue cattle are famous for their exceptional muscular development or "double-muscling". This defining feature emerged following the fixation of a loss-of-function variant in the myostatin gene in the eighties. Since then, sustained selection has further increased muscle mass of Belgian Blue animals to a comparable extent. In the present paper, we study the genetic determinants of this second wave of muscle growth. RESULTS: A scan for selective sweeps did not reveal the recent fixation of another allele with major effect on muscularity. However, a genome-wide association study identified two genome-wide significant and three suggestive quantitative trait loci (QTL) affecting specific muscle groups and jointly explaining 8-21% of the heritability. The top two QTL are caused by presumably recent mutations on unique haplotypes that have rapidly risen in frequency in the population. While one appears on its way to fixation, the ascent of the other is compromised as the likely underlying MRC2 mutation causes crooked tail syndrome in homozygotes. Genomic prediction models indicate that the residual additive variance is largely polygenic. CONCLUSIONS: Contrary to complex traits in humans which have a near-exclusive polygenic architecture, muscle mass in beef cattle (as other production traits under directional selection), appears to be controlled by (i) a handful of recent mutations with large effect that rapidly sweep through the population, and (ii) a large number of presumably older variants with very small effects that rise slowly in the population (polygenic adaptation)

Prednisolone restores blood brain barrier damages in dystrophic MDX mouse

Although the glucocorticoids delay the progression of Duchenne muscular dystrophy (DMD) their mechanism of action is unknown. In our previous studies we demonstrated that in the mdx mice, an animal model of DMD, besides the muscle degeneration, serious damages of the blood-brain barrier (BBB) occur taking to enhanced vessels permeability and brain edema (1). Moreover, we observed that the mdx mice after α–methyl-prednisolone (PDN) treatment ameloriated the histopathological profiles and the excitation-contraction of the myofibers (2). In this study, we evaluated the effects of the PDN on the BBB of the mdx mice, by estimating the immunocytochemical and biochemical expression of endothelial ZO-1 and occludin, pericyte desmin, and glial GFAP and short dystrophin isoform Dp 71 proteins, used as BBB markers. In addition, we analyzed the expression of dystrophin associate proteins (DAPs) aquaporin-4 (AQP4) and α-β dystroglycan in parallel in both brain and muscles of PDN treated mdx as well as in control mice. Results showed in mdx PDN treated mice a significant increase of the mRNA and protein content of all the glial, pericyte and endothelial proteins as compared to untreated mdx. Moreover, by immunoprecipitation we demonstrated that the BBB alteration in the mdx mice were coupled with enhanced occludin and AQP4 phosphorylation degree which, instead, was reduced after PDN treatment. Finally we observed that AQP4 and α-β dystroglycan complex increases its mRNA and protein content in both PDN mdx brain and muscle fibers, compared with mdx mice where the perivascular glial membranes and the myofibers showed a light staining after immunofluorescence analysis . These data indicate that the PDN restores the BBB damages in the mdx mice by inducing in the glial cell the expression of GFAP, AQP4 and Dp71 proteins and in the pericytes and endothelial cells, of the desmin and ZO-1 proteins, which are deficient in the distrophic mice. Moreover, the reduction in the AQP4 and occludin phosphorylation degree coupled with their ankoring to glial and endothelial membranes in the PDN mdx mice suggests that the glial and endothelial cells may be a cellular target of the drug. Finally, the enhanced expression of DAPs AQP4 and α-β dystroglycan in both brain and myofibers of PDN treated mdx mice compared to untreated mdx ones suggest the PDN might ameliorate the brain vessels and muscles functions of the dystrophic mice by a restoring a correct links between DAPs proteins and the extracellular matrix. 1. Nico B et al. Glia, 42: 235-251. (2003). 2. Cozzoli A. et al., Neuropathol. Appl. Neurobiol. 37, 243-256 (2011)

Generation of Induced Pluripotent Stem Cells from Patients with Duchenne Muscular Dystrophy and their induction to Neurons

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by deficient expression of the cytoskeletal protein dystrophin. DMD has been associated with intellectual disability and mental retardation (MR) and is present in about a third of all patients. Loss of Dp71, the major dystrophin-gene product in brain, and the dystrophin associated proteins (DAPs) are thought to contribute to severity of MR, but the specific function of the neural dystrophin proteins are poorly understood for a limited access to DMD patients brain tissue (1). Differentiation of induced Pluripotent Stem Cells (iPSCs) provides an opportunity to generate an unlimited supply of living neurons genetically identical to those present in patients. In this study we obtained DMD-iPSCs from peripheral blood mononuclear cells of DMD patients with cognitive impairment and we performed morphological (fluorescence and electron microscopy), molecular (Western Blot and Real Time PCR) and functional (electrophysiology) characterization both of iPSC-derived Neural Stem Cells (NSCs) and the differentiated neurons. Preliminary data showed a reduction of Dp71 and DAPs proteins, including the AQP4, potassium channel Kir4.1, α- and β-dystroglycan (α/βDG) and α-syntrophin (αSyn), both at transcriptional and traductional level, coupled with membrane dys-arrangment in DMD-iPSCs compared with healthy iPSCs. Moreover, we demonstrated that the neurons obtained from the differentiation of iPSCs derived from DMD patient showed after confocal analysis, altered cytoskeleton and reduction in Dp71expression, and by single-cell imaging experiments and electrophysiology, altered intracellular calcium homeostasis, in analogy with what shown in the dystrophic mdx mouse neurons (2). Overall these results showed that the Dp71 and DAPs alterations affect also the neural precursor as well as the differentiated neurons in DMD patients, so suggesting a key role in the pathogenesis of neurocognitive deficits in DMD disease

Balancing Selection of a Frame-Shift Mutation in the MRC2 Gene Accounts for the Outbreak of the Crooked Tail Syndrome in Belgian Blue Cattle

We herein describe the positional identification of a 2-bp deletion in the open reading frame of the MRC2 receptor causing the recessive Crooked Tail Syndrome in cattle. The resulting frame-shift reveals a premature stop codon that causes nonsense-mediated decay of the mutant messenger RNA, and the virtual absence of functional Endo180 protein in affected animals. Cases exhibit skeletal anomalies thought to result from impaired extracellular matrix remodeling during ossification, and as of yet unexplained muscular symptoms. We demonstrate that carrier status is very significantly associated with desired characteristics in the general population, including enhanced muscular development, and that the resulting heterozygote advantage caused a selective sweep which explains the unexpectedly high frequency (25%) of carriers in the Belgian Blue Cattle Breed

Comparing Pandemic to Seasonal Influenza Mortality: Moderate Impact Overall but High Mortality in Young Children

Background: We assessed the severity of the 2009 influenza pandemic by comparing pandemic mortality to seasonal influenza mortality. However, reported pandemic deaths were laboratory-confirmed - and thus an underestimation - whereas seasonal influenza mortality is often more inclusively estimated. For a valid comparison, our study used the same statistical methodology and data types to estimate pandemic and seasonal influenza mortality. Methods and Findings: We used data on all-cause mortality (1999-2010, 100% coverage, 16.5 million Dutch population) and influenza-like-illness (ILI) incidence (0.8% coverage). Data was aggregated by week and age category. Using generalized estimating equation regression models, we attributed mortality to influenza by associating mortality with ILI-incidence, while adjusting for annual shifts in association. We also adjusted for respiratory syncytial virus, hot/cold weather, other seasonal factors and autocorrelation. For the 2009 pandemic season, we estimated 612 (range 266-958) influenza-attributed deaths; for seasonal influen