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Differentiation and Contractile Analysis of GFP-Sarcomere Reporter hiPSC-Cardiomyocytes.
Authors: Sharma A, Toepfer CN, Schmid M, Garfinkel AC, Seidman CE Abstract Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful cellular platform for illuminating mechanisms of human cardiovascular disease and for pharmacological screening. Recent advances in CRISPR/Cas9-mediated genome editing technology underlie this profound utility. We have generated hiPSC-CMs harboring fluorescently-tagged sarcomeric proteins, which provide a tool to non-invasively study human sarcomere function and dysfunction. In this unit, we illustrate methods for conducting high-efficiency, ...
Source: Current Protocols in Human Genetics - January 25, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Matchmaker Exchange.
This article guides usage of the MME for rare disease gene discovery. © 2017 by John Wiley & Sons, Inc. PMID: 29044468 [PubMed - in process] (Source: Current Protocols in Human Genetics)
Source: Current Protocols in Human Genetics - October 20, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Co-Differentiation of Human Pluripotent Stem Cells-Derived Cardiomyocytes and Endothelial Cells from Cardiac Mesoderm Provides a Three-Dimensional Model of Cardiac Microtissue.
Authors: Giacomelli E, Bellin M, Orlova VV, Mummery CL Abstract The formation of cardiac mesodermal subtypes is highly regulated in time and space during heart development. In vitro models based on human pluripotent stem cells (hPS cells) provide opportunities to study mechanisms underlying fate choices governing lineage specification from common cardiovascular progenitors in human embryos. The generation of cardiac endothelial cells in particular allows the creation of complex models of cardiovascular disorders in which either cardiomyocytes or endothelial cells are affected. Here, a protocol for co-diffe...
Source: Current Protocols in Human Genetics - October 20, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Analysis of Gene-Gene Interactions.
Authors: Cole BS, Hall MA, Urbanowicz RJ, Gilbert-Diamond D, Moore JH Abstract The goal of this unit is to introduce epistasis, or gene-gene interactions, as a significant contributor to the genetic architecture of complex traits, including disease susceptibility. This unit begins with an historical overview of the concept of epistasis and the challenges inherent in the identification of potential gene-gene interactions. Then, it reviews statistical and machine learning methods for discovering epistasis in the context of genetic studies of quantitative and categorical traits. This unit concludes with a dis...
Source: Current Protocols in Human Genetics - October 20, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Analysis and Annotation of Whole-Genome or Whole-Exome Sequencing Derived Variants for Clinical Diagnosis.
Authors: Worthey EA Abstract Over the last 10 years, next-generation sequencing (NGS) has transformed genomic research through substantial advances in technology and reduction in the cost of sequencing, and also in the systems required for analysis of these large volumes of data. This technology is now being used as a standard molecular diagnostic test in some clinical settings. The advances in sequencing have come so rapidly that the major bottleneck in identification of causal variants is no longer the sequencing or analysis (given access to appropriate tools), but rather clinical interpretation. Interpr...
Source: Current Protocols in Human Genetics - October 20, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Population Stratification in Genetic Association Studies.
Authors: Hellwege JN, Keaton JM, Giri A, Gao X, Velez Edwards DR, Edwards TL Abstract Population stratification (PS) is a primary consideration in studies of genetic determinants of human traits. Failure to control for PS may lead to confounding, causing a study to fail for lack of significant results, or resources to be wasted following false-positive signals. Here, historical and current approaches for addressing PS when performing genetic association studies in human populations are reviewed. Methods for detecting the presence of PS, including global and local ancestry methods, are described. Also descr...
Source: Current Protocols in Human Genetics - October 20, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Induced Pluripotent Stem Cells from Ovarian Tissue.
Authors: Salas S, Ng N, Gerami-Naini B, Anchan RM Abstract Yamanaka and colleagues revolutionized stem cell biology and regenerative medicine by observing that somatic cells can be reprogrammed into pluripotent stem cells. Evidence indicates that induced pluripotent stem (iPS) cells retain epigenetic memories that bias their spontaneous differentiation into the originating somatic cell type, therefore epigenetic memory may be exploited to improve tissue specific regeneration. We recently showed that iPS cells reprogrammed from ovarian granulosa cells using mouse and human tissue overwhelmingly differentiat...
Source: Current Protocols in Human Genetics - October 20, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Copy-Number Variants Detection by Low-Pass Whole-Genome Sequencing.
Authors: Dong Z, Xie W, Chen H, Xu J, Wang H, Li Y, Wang J, Chen F, Choy KW, Jiang H Abstract Emerging studies have demonstrated that whole-genome sequencing (WGS) is an efficient tool for copy-number variants (CNV) detection, particularly in probe-poor regions, as compared to chromosomal microarray analysis (CMA). However, the cost of testing is beyond economical for routine usage and the lengthy turn-around time is not ideal for clinical implementation. In addition, the demand for computational resources also reduces the probability of clinical integration into each laboratory. Herein, a protocol providi...
Source: Current Protocols in Human Genetics - July 12, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

1D Genome Sequencing on the Oxford Nanopore MinION.
Authors: Goodwin S, Wappel R, McCombie WR Abstract Today's short-read sequencing instruments can generate read lengths between 50 bp and 700 bp depending on the specific instrument. These high-throughput sequencing approaches have revolutionized genomic science, allowing hundreds of thousands of full genomes to be sequenced, and have become indispensable tools for many researchers. With greater insight has come the revelation that many genomes are much more complicated than originally thought and include many rearrangements and copy-number variations. Unfortunately, short-read sequencing technologies are n...
Source: Current Protocols in Human Genetics - July 12, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Microscopy and Image Analysis.
Authors: McNamara G, Difilippantonio M, Ried T, Bieber FR Abstract This unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and fluorescence in situ hybridization (FISH). We believe there are excellent opportunities for cytogeneticists, pathologists, and other biomedical readers, to take advantage of specimen optical clearing techniques and expansion microscopy-we briefly point to these new opportunities. © 2017 by John Wiley & Sons, Inc. PMID: 28696557 [PubMed - in process] (Source: Current Protoc...
Source: Current Protocols in Human Genetics - July 12, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Mammalian Cell Tissue Culture.
Authors: Phelan K, May KM Abstract Cultured mammalian cells are used extensively in the field of human genetics. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2017 by John Wiley & Sons, Inc. PMID: 28696558 [PubMed - in process] (Source: Current...
Source: Current Protocols in Human Genetics - July 12, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Genetic Testing for Hereditary Nonpolyposis Colorectal Cancer (HNPCC).
Authors: Nallamilli BRR, Hegde M Abstract Hereditary nonpolyposis colorectal cancer (HNPCC), also called Lynch syndrome, is an autosomal dominant cancer syndrome that confers an elevated risk of early-onset colorectal cancer (CRC) and increased lifetime risk for other cancers of the endometrium, stomach, small intestine, hepatobiliary system, kidney, ureter, and ovary. Lynch syndrome accounts for up to 3% of all CRC, making it the most common hereditary colorectal cancer syndrome. Germline mutations in methyl-directed mismatch repair (MMR) genes give rise to microsatellite instability (MSI) in tumor DNA. L...
Source: Current Protocols in Human Genetics - July 12, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Overview of Admixture Mapping.
Authors: Shriner D Abstract Admixture mapping is a powerful method of gene mapping for diseases or traits that show differential risk by ancestry. Admixture mapping has been applied most often to Americans who trace ancestry to various combinations of Native Americans, Europeans, and West Africans. Recent developments in admixture mapping include improvements in methods and the reference data needed to make inferences about ancestry, as well as extensions of the mapping approach in the framework of linear mixed models. In this unit, the key concepts of admixture mapping are outlined. Several approaches for...
Source: Current Protocols in Human Genetics - July 12, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

High-Risk Screening for Fabry Disease: Analysis by Tandem Mass Spectrometry of Globotriaosylceramide (Gb3 ) in Urine Collected on Filter Paper.
Authors: Auray-Blais C, Lavoie P, Boutin M, Abaoui M Abstract Fabry disease is a complex, panethnic lysosomal storage disorder. It is characterized by the accumulation of glycosphingolipids in tissues, organs, the vascular endothelium, and biological fluids. The reported incidence in different populations is quite variable, ranging from 1:1400 to 1:117,000. Its complexity lies in the marked genotypic and phenotypic heterogeneity. Despite the fact that it is an X-linked disease, more than 600 mutations affect both males and females. In fact, some females may be affected as severely as males. The purpose of ...
Source: Current Protocols in Human Genetics - April 7, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Methods for Quantitative Creatinine Determination.
Authors: Moore JF, Sharer JD Abstract Reliable measurement of creatinine is necessary to assess kidney function, and also to quantitate drug levels and diagnostic compounds in urine samples. The most commonly used methods are based on the Jaffe principal of alkaline creatinine-picric acid complex color formation. However, other compounds commonly found in serum and urine may interfere with Jaffe creatinine measurements. Therefore, many laboratories have made modifications to the basic method to remove or account for these interfering substances. This appendix will summarize the basic Jaffe method, as well ...
Source: Current Protocols in Human Genetics - April 7, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research