Current Protocols in Human Genetics最新文献

Fabry disease is an X-linked lysosomal storage disorder, caused by a deficit in α-galactosidase A enzyme activity, leading to the storage of sphingolipids such as globotriaosylsphingosine (lyso-Gb₃), globotriaosylceramide (Gb₃), and galabiosylceramide (Ga₂) in organs, tissues and biological fluids. A recent metabolomic study performed in plasma revealed lyso-Gb₃ analogs as novel Fabry disease biomarkers. These molecules correspond to lyso-Gb₃ with different chemical modifications on the sphingosine chain (−C₂H₄, −H₂, +O, +H₂O, +H₂O_2, and +H₂O₃). An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the multiplex analysis of lyso-Gb₃ and its 6 analogs in plasma. The samples are prepared by solid phase extraction using mixed-mode strong cation exchange (MCX) cartridges. An in-house synthesized N-glycinated lyso-Gb₃ derivative was used for the internal standard. The limits of detection (LODs) measured for lyso-Gb₃ and its analogs ranged from 0.06 to 0.29 nM. © 2016 by John Wiley & Sons, Inc.

The R package epigenomix has been designed to detect differentially transcribed gene isoforms that, in addition, exhibit altered histone modifications at their respective genomic loci. The package provides methods to map histone ChIP-seq profiles to isoforms and estimate their transcript abundances from RNA-seq data. Based on the differences observed between case and control samples in the RNA-seq and ChIP-seq data, a correlation measure is calculated for each isoform. The distribution of this correlation measure is further investigated by a Bayesian mixture model to (i) reveal the relationship between the studied histone modification and transcriptional activity, and (ii) detect specific isoforms with differences in both transcription values and histone modifications. The method is designed for experiments with a few or no replicates, and is superior to separate analyses of both data types in that setting. This unit illustrates the integrative analysis of ChIP-seq and RNA-seq data with epigenomix. © 2016 by John Wiley & Sons, Inc.

The hypothalamus comprises neuronal clusters that are essential for body weight regulation and other physiological functions. Insights into the complex cellular physiology of this region of the brain are critical to understanding the pathogenesis of obesity, but human hypothalamic cells are largely inaccessible for direct study. Here we describe a technique for generation of arcuate-like hypothalamic neurons from human pluripotent stem (hPS) cells. Early activation of SHH signaling and inhibition of BMP and TGFβ signaling, followed by timed inhibition of NOTCH, can efficiently differentiate hPS cells into NKX2.1+ hypothalamic progenitors. Subsequent incubation with BDNF induces the differentiation and maturation of pro-opiomelanocortin and neuropeptide Y neurons, which are major cell types in the arcuate hypothalamus. These neurons have molecular and cellular characteristics consistent with arcuate neurons. © 2016 by John Wiley & Sons, Inc.

The Illumina HumanExome BeadChip and other exome-based genotyping arrays offer inexpensive genotyping of some 240,000 mostly nonsynonymous coding variants across the human genome. The HumanExome chip, with its highly non-uniform distribution of markers and emphasis on rare coding variants, presents some unique challenges for quality control (QC) and data cleaning. Here, we describe QC procedures for HumanExome data, with examples of challenges specific to exome arrays from our experience cleaning a data set of ∼7,500 samples from the NEIGHBORHOOD Consortium. We focus on standard procedures for QC of genome-wide array data including genotype calling, sex verification, sample identity verification, relationship checking, and population structure that are complicated by the HumanExome panel's enrichment in rare, exonic variation. © 2016 by John Wiley & Sons, Inc.

Animal models are an important resource for studying human diseases. Genetically engineered mice are the most commonly used species and have made significant contributions to our understanding of basic biology, disease mechanisms, and drug development. However, they often fail to recreate important aspects of human diseases and thus can have limited utility as translational research tools. Developing disease models in species more similar to humans may provide a better setting in which to study disease pathogenesis and test new treatments. This unit provides an overview of the history of genetically engineered large animals and the techniques that have made their development possible. Factors to consider when planning a large animal model, including choice of species, type of modification and methodology, characterization, production methods, and regulatory compliance, are also covered. © 2016 by John Wiley & Sons, Inc.

Chromosome analysis is one of the first approaches to genetic testing and remains a key component of genetic analysis of constitutional and somatic genetic disorders. Numerical or unbalanced structural chromosome abnormalities usually lead to multiple congenital anomalies. Sometimes these are compatible with live birth, usually resulting in severe cognitive and physical handicaps; other times they result in miscarriage or stillbirth. Chromosome rearrangements also occur as somatic changes in malignancies. Identification of constitutional chromosomal anomalies (anomalies present in most or all cells of the body and/or the germline) can provide important information for genetic counseling. In this unit, we introduce chromosomal microarray analysis (CMA), which is a relatively recent addition to cytogenetic technologies, and has become the recommended first-tier testing method for patients with developmental delay, intellectual disability, autism, and/or multiple congenital anomalies. We also discuss non-invasive prenatal testing/screening (NIPTS), which uses circulating cell-free fetal DNA (cfDNA) from maternal plasma to rapidly screen for autosomal and sex-chromosome aneuploidies. Cytogenetic analysis of tumors is helpful in diagnosis and in monitoring the effects of treatment. The protocols in this chapter cover the clinical study of chromosomes in nonmalignant tissues. © 2016 by John Wiley & Sons, Inc.