High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling

Human individuals differ from one another at only ~0.1% of nucleotide positions, but these single nucleotide differences account for most heritable phenotypic variation. Large-scale efforts to discover and genotype human variation have been limited to common polymorphisms. However, these efforts overlook rare nucleotide changes that may contribute to phenotypic diversity and genetic disorders, including cancer. Thus, there is an increasing need for high-throughput methods to robustly detect rare nucleotide differences. Toward this end, we have adapted the mismatch discovery method known as Ecotilling for the discovery of human single nucleotide polymorphisms. To increase throughput and reduce costs, we developed a universal primer strategy and implemented algorithms for automated band detection. Ecotilling was validated by screening 90 human DNA samples for nucleotide changes in 5 gene targets and by comparing results to public resequencing data. To increase throughput for discovery of rare alleles, we pooled samples 8-fold and found Ecotilling to be efficient relative to resequencing, with a false negative rate of 5% and a false discovery rate of 4%. We identified 28 new rare alleles, including some that are predicted to damage protein function. The detection of rare damaging mutations has implications for models of human disease.     

Spreading of silent chromatin: inaction at a distance

One of the oldest unsolved problems in genetics is the observation that gene silencing can 'spread' along a chromosome. Although spreading has been widely perceived as a process of long-range assembly of heterochromatin proteins, such 'oozing' might not apply in most cases. Rather, long-range silencing seems to be a dynamic process, involving local diffusion of histone-modifying enzymes from source binding sites to low-affinity sites nearby. Discontinuous silencing might reflect looping interactions, whereas the spreading of continuous silencing might be driven by the processive movement of RNA or DNA polymerases. We review the evidence for the spreading of silencing in many contexts and organisms and conclude that multiple mechanisms have evolved that silence genes at a distance.     

"Point" centromeres of Saccharomyces harbor single centromere-specific nucleosomes

The "point" centromere of budding yeast is genetically defined by an ≈ 125-bp sequence. Recent fluorescence measurements of kinetochore clusters have suggested that this sequence specifies multiple centromere histone 3 (CenH3) nucleosomes. However, high-resolution mapping demonstrates that there is only one CenH3 nucleosome per centromere, providing biochemical confirmation of the point centromere model.     

Sixty years of genome biology

Sixty years after Watson and Crick published the double helix model of DNA''s structure, thirteen members of Genome Biology''s Editorial Board select key advances in the field of genome biology subsequent to that discovery.April 25th 2013 is the sixtieth anniversary of the infamous Watson and Crick Nature paper describing a model for the structure of DNA, published 25 April 1953: the now infamous ''double helix'' [1]. Two accompanying papers from Rosalind Franklin, Maurice Wilkins and colleagues leant experimental support to the proposed structure in the form of X-ray diffraction data [2,3], as described elsewhere in this issue of Genome Biology [4]. The model was a landmark discovery in the history of modern science, and was notable for its cross-disciplinary importance: the question addressed was of immense biological importance, but it was physicists and chemists whose expertise and techniques were needed in order to arrive at an answer. One of these physicists, Ray Gosling, describes the unveiling of Watson and Crick''s double helix structure as a ''eureka'' moment [4]: its simplicity and elegance were striking, and not only explained the X-ray diffraction data but also the mode of replication of life itself. It is rare for a scientific discovery to achieve such an iconic status, to pervade popular culture and the public consciousness, as well as to become an emblem of scientific inquiry - as exemplified by Genome Biology''s double helix-inspired logo. Although Avery had already shown DNA to be the genetic material [5], it took the convincing simplicity of Watson and Crick''s double helix for this notion to widely take hold, in place of theories favoring proteins. The discovery, therefore, had many important implications, and set the scene for future breakthroughs in the field of genome biology.To celebrate sixty years of such discoveries, we asked a jury composed of Genome Biology Editorial Board members to select key advances in the field since 25 April 1953. The brief was to choose a development that was either the most important or the most surprising, or that had the most personal impact, and to briefly summarize why. A number of selections focused on technological advances - from restriction mapping through microarrays and high-throughput sequencing. These technologies have clearly done much to inform our understanding of the biology of genomes. The most popular choice, however, was the discovery of introns. Much like the double helix, this discovery had something of the ''X factor'' to it: biologists trained in the post-intron era may take the concept of gene fragmentation for granted, but at the time it was a truly radical and paradigm-shifting idea. The sense of surprise made a strong impression on those old enough to remember the discovery, and one of the groups involved went so far as to describe it as ''amazing'' in the title of their paper [6].     

A blood based 12-miRNA signature of Alzheimer disease patients

Background

Alzheimer disease (AD) is the most common form of dementia but the identification of reliable, early and non-invasive biomarkers remains a major challenge. We present a novel miRNA-based signature for detecting AD from blood samples.

Results

We apply next-generation sequencing to miRNAs from blood samples of 48 AD patients and 22 unaffected controls, yielding a total of 140 unique mature miRNAs with significantly changed expression levels. Of these, 82 have higher and 58 have lower abundance in AD patient samples. We selected a panel of 12 miRNAs for an RT-qPCR analysis on a larger cohort of 202 samples, comprising not only AD patients and healthy controls but also patients with other CNS illnesses. These included mild cognitive impairment, which is assumed to represent a transitional period before the development of AD, as well as multiple sclerosis, Parkinson disease, major depression, bipolar disorder and schizophrenia. miRNA target enrichment analysis of the selected 12 miRNAs indicates an involvement of miRNAs in nervous system development, neuron projection, neuron projection development and neuron projection morphogenesis. Using this 12-miRNA signature, we differentiate between AD and controls with an accuracy of 93%, a specificity of 95% and a sensitivity of 92%. The differentiation of AD from other neurological diseases is possible with accuracies between 74% and 78%. The differentiation of the other CNS disorders from controls yields even higher accuracies.

Conclusions

The data indicate that deregulated miRNAs in blood might be used as biomarkers in the diagnosis of AD or other neurological diseases.     

Ovariectomy and overeating palatable, energy-dense food increase subcutaneous adipose tissue more than intra-abdominal adipose tissue in rats

Background

Men are at an increased risk of dying from heart failure caused by inflammatory heart diseases such as atherosclerosis, myocarditis and dilated cardiomyopathy (DCM). We previously showed that macrophages in the spleen are phenotypically distinct in male compared to female mice at 12 h after infection. This innate immune profile mirrors and predicts the cardiac immune response during acute myocarditis.

Methods

In order to study sex differences in the innate immune response, five male and female BALB/c mice were infected intraperitoneally with coxsackievirus B3 (CVB3) or phosphate buffered saline and their spleens were harvested 12 h later for microarray analysis. Gene expression was determined using an Affymetrix Mouse Gene 1.0 ST Array. Significant gene changes were verified by quantitative real-time polymerase chain reaction or ELISA.

Results

During the innate immune response to CVB3 infection, infected males had higher splenic expression of genes which are important in regulating the influx of cholesterol into macrophages, such as phospholipase A₂ (PLA₂) and the macrophage scavenger receptor compared to the infected females. We also observed a higher expression in infected males compared to infected females of squalene synthase, an enzyme used to generate cholesterol within cells, and Cyp2e1, an enzyme important in metabolizing cholesterol and steroids. Infected males also had decreased levels of the translocator protein 18 kDa (TSPO), which binds PLA₂ and is the rate-limiting step for steroidogenesis, as well as decreased expression of the androgen receptor (AR), which indicates receptor activation. Gene differences were not due to increased viral replication, which was unaltered between sexes.

Conclusions

We found that, compared to females, male mice had a greater splenic expression of genes which are important for cholesterol metabolism and activation of the AR at 12 h after infection. Activation of the AR has been linked to increased cardiac hypertrophy, atherosclerosis, myocarditis/DCM and heart failure in male mice and humans.