Current Protocols in Human Genetics Current Protocols in Human Genetics RSS feedThis is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.

Quantification of glycosaminoglycans in urine by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry.
Authors: Zhang H, Young SP, Millington DS Abstract Mucopolysaccharidoses (MPSs) are complex lysosomal storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are disease-specific biomarkers for MPSs. This unit describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic or iduronic ...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

A multifaceted FISH approach to study endogenous RNAs and DNAs in native nuclear and cell structures.
Authors: Byron M, Hall LL, Lawrence JB Abstract Fluorescence in situ hybridization (FISH) is not a singular technique, but a battery of powerful and versatile tools for examining the distribution of endogenous genes and RNAs in precise context with each other and in relation to specific proteins or cell structures. This unit offers the details of highly sensitive and successful protocols that were initially developed largely in our lab and honed over a number of years. Our emphasis is on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre-mRNA metabolism, ...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Predicting functional effect of human missense mutations using PolyPhen-2.
Authors: Adzhubei I, Jordan DM, Sunyaev SR Abstract PolyPhen-2 (Polymorphism Phenotyping v2), available as software and via a Web server, predicts the possible impact of amino acid substitutions on the stability and function of human proteins using structural and comparative evolutionary considerations. It performs functional annotation of single-nucleotide polymorphisms (SNPs), maps coding SNPs to gene transcripts, extracts protein sequence annotations and structural attributes, and builds conservation profiles. It then estimates the probability of the missense mutation being damaging based on a combinati...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Overview of molecular genetic diagnosis.
Authors: Harada S, Korf BR Abstract This unit describes the overview and strategies for molecular genetic diagnosis. Molecular genetic testing is typically used to detect changes in a single gene, and includes direct sequence analysis and detection of large deletion/duplication. Occasionally linkage analysis is still used when the gene is not known. Direct mutation analysis is possible if the gene responsible for a disorder has been identified. Genetic linkage analysis is used when polymorphic markers are available that are closely linked to a disease gene but the gene itself has not been cloned, or when i...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Methods for meta-analysis of genetic data.
Authors: Lunetta KL Abstract Modern genetic association studies, using genome-wide genotype data, are often underpowered. Meta-analyses of multiple studies performing genome-wide genotyping improve power and have led to the identification of thousands of genotype-trait associations. This unit provides an overview of the key concepts required for genetic meta-analyses, and presents strategic approaches and key decisions that must be made in the process of performing genome-wide association study (GWAS) meta-analyses. The commentary discusses the interpretation of GWAS meta-analysis results, complications, a...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Diagnosing lysosomal storage disorders: Fabry disease.
Authors: Bodamer OA, Johnson B, Dajnoki A Abstract Fabry disease (FD) is an X-linked lysosomal storage disorder due to deficiency of alpha galactosidase A (GLA). Progressive, intralysosomal accumulation of neutral glycosphingolipids in endothelial cells and podocytes leads to multi-organ involvement in affected males and to a lesser extent in affected females. Diagnosis of FD is based on GLA analysis in leukocytes or dried blood spots (DBS) in FD males while GLA activities may be within the normal range in FD females. The advent of fluorometric and mass spectrometry methods for enzyme analysis in DBS has s...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Analysis of epigenetic modifications of DNA in human cells.
Authors: Kristensen LS, Treppendahl MB, Grønbæk K Abstract Epigenetics, the study of somatically heritable changes in gene expression not related to changes in the DNA sequence, is a rapidly expanding research field that plays important roles in healthy as well as in diseased cells. DNA methylation and hydroxymethylation are epigenetic modifications found in human cells, which are deeply implicated in normal cellular processes as well as in several major human diseases. Here, a range of different methods for the analyses of DNA methylation and hydroxymethylation at locus-specific and genome-wide scales i...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Whole genome sequencing: a considered approach to clinical implementation.
Authors: Dimmock D Abstract The recent entry of "whole" exome/"whole" genome sequencing into limited clinical practice has led to a progression of the availability of genome-scale testing beyond deletion/duplication copy number arrays. This unit provides a considered approach to the implementation of such testing in routine clinical practice. Specifically, we will highlight the challenges in patient selection and consent, and the technical issues surrounding test interpretation and reporting. The unit will then provide practical solutions that allow for genome-wide sequencing to be implemented in current c...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Management of incidental findings in clinical genomic sequencing.
Authors: Krier JB, Green RC Abstract Genomic sequencing is becoming accurate, fast, and inexpensive, and is rapidly being incorporated into clinical practice. Incidental findings, which result in large numbers from genomic sequencing, are a potential barrier to the utility of this new technology due to their high prevalence and the lack of evidence or guidelines available to guide their clinical interpretation. This unit reviews the definition, classification, and management of incidental findings from genomic sequencing. The unit focuses on the clinical aspects of handling incidental findings, with an emp...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Genotype imputation in genome-wide association studies.
Authors: Porcu E, Sanna S, Fuchsberger C, Fritsche LG Abstract Imputation is an in silico method that can increase the power of association studies by inferring missing genotypes, harmonizing data sets for meta-analyses, and increasing the overall number of markers available for association testing. This unit provides an introductory overview of the imputation method and describes a two-step imputation approach that consists of the phasing of the study genotypes and the imputation of reference panel genotypes into the study haplotypes. Detailed steps for data preparation and quality control illustrate how ...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Identifying rare variants associated with complex traits via sequencing.
Authors: Li B, Liu DJ, Leal SM Abstract Although genome-wide association studies have been successful in detecting associations with common variants, there is currently an increasing interest in identifying low-frequency and rare variants associated with complex traits. Next-generation sequencing technologies make it feasible to survey the full spectrum of genetic variation in coding regions or the entire genome. The association analysis for rare variants is challenging, and traditional methods are ineffective, however, due to the low frequency of rare variants, coupled with allelic heterogeneity. Recently...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Oncolytic adenoviruses: design, generation, and experimental procedures.
Authors: Davydova J, Yamamoto M Abstract Oncolytic adenoviruses are designed to take advantage of the virus' native ability to replicate in cancer cells to induce oncolysis. Subsequently, the released viral progeny spread and kill the neighboring cancer cells. These characteristics, together with the ability of adenovirus to infect a broad spectrum of cells, its well understood replication machinery, and relative ease of manufacture have led to the intensive use of adenovirus as an anticancer agent. This unit describes cloning strategies, procedures to turn the intended design into virus, and quality analy...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Gene delivery to the airway.
Authors: Keiser NW, Engelhardt JF Abstract This unit describes generation of and gene transfer to several commonly used airway models. Isolation and transduction of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined. Transduction of these polarized cells is also described. Methods are presented for generation of tracheal xenografts, as well as both ex vivo and in vivo gene transfer to these xenografts. Finally, a method for in vivo gene delivery to the lungs of rodents is included. Methods for evaluating transgene expression are ...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Single nucleotide polymorphism genotyping using BeadChip microarrays.
Authors: Lambert G, Tsinajinnie D, Duggan D Abstract The genotyping of single nucleotide polymorphisms (SNPs) has successfully contributed to the study of complex diseases more than any other technology to date. Genome-wide association studies (GWAS) using 10,000s to >1,000,000 SNPs have identified 1000s of statistically significant SNPs pertaining to 17 different human disease and trait categories. Post-GWAS fine-mapping studies using 10,000s to 100,000s SNPs on a microarray have narrowed the region of interest for many of these GWAS findings; in addition, independent signals within the original GWAS r...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Analyzing copy number variation using SNP array data: protocols for calling CNV and association tests.
Authors: Lin CF, Naj AC, Wang LS Abstract High-density SNP genotyping technology provides a low-cost, effective tool for conducting Genome Wide Association (GWA) studies. The wide adoption of GWA studies has indeed led to discoveries of disease- or trait-associated SNPs, some of which were subsequently shown to be causal. However, the nearly universal shortcoming of many GWA studies--missing heritability--has prompted great interest in searching for other types of genetic variation, such as copy number variation (CNV). Certain CNVs have been reported to alter disease susceptibility. Algorithms and tools ha...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research