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Reporting of Diagnostic Cytogenetic Results.
Authors: Giersch AB, Bieber FR, Dubuc AM, Fletcher JA, Ligon AH, Mason-Suares H, Morton CC, Weremowicz S, Xiao S, Cin PD Abstract This appendix, developed by the staff at the Center for Advanced Molecular Diagnostics in the Department of Pathology at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a useful reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis ty...
Source: Current Protocols in Human Genetics - April 3, 2016 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

GONAD: A Novel CRISPR/Cas9 Genome Editing Method that Does Not Require Ex Vivo Handling of Embryos.
Authors: Gurumurthy CB, Takahashi G, Wada K, Miura H, Sato M, Ohtsuka M Abstract Transgenic technologies used for creating a desired genomic change in animals involve three critical steps: isolation of fertilized eggs, microinjection of transgenic DNA into them and their subsequent transfer to recipient females. These ex vivo steps have been widely used for over 3 decades and they were also readily adapted for the latest genome editing technologies such as ZFNs, TALENs, and CRISPR/Cas9 systems. We recently developed a method called GONAD (Genome editing via Oviductal Nucleic Acids Delivery) that does not r...
Source: Current Protocols in Human Genetics - January 11, 2016 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Efficient CRISPR/Cas9-Based Genome Engineering in Human Pluripotent Stem Cells.
Authors: Kime C, Mandegar MA, Srivastava D, Yamanaka S, Conklin BR, Rand TA Abstract Human pluripotent stem cells (hPS cells) are rapidly emerging as a powerful tool for biomedical discovery. The advent of human induced pluripotent stem cells (hiPS cells) with human embryonic stem (hES)-cell-like properties has led to hPS cells with disease-specific genetic backgrounds for in vitro disease modeling and drug discovery as well as mechanistic and developmental studies. To fully realize this potential, it will be necessary to modify the genome of hPS cells with precision and flexibility. Pioneering experiments...
Source: Current Protocols in Human Genetics - January 11, 2016 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

In Silico Functional Annotation of Genomic Variation.
Authors: Butkiewicz M, Bush WS Abstract This unit describes the concepts and practical techniques for annotating genomic variants in the human genome to estimate their functional significance. With the rapid increase of available whole exome and whole genome sequencing information for human studies, annotation techniques have become progressively more important for highlighting and prioritizing nucleotide variants and their potential impact on genes and other genetic constructs. Here, we present an overview of different types of variant annotation approaches and elaborate on their foundations, assumptions,...
Source: Current Protocols in Human Genetics - January 11, 2016 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Spinal Muscular Atrophy: Overview of Molecular Diagnostic Approaches.
Authors: Prior TW, Nagan N Abstract Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease and the most common genetic cause of infant mortality, affecting ∼1 in 10,000 live births. The disease is characterized by progressive symmetrical muscle weakness resulting from the degeneration and loss of anterior horn cells in the spinal cord and brain stem nuclei. The disease is classified on the basis of age of onset and clinical course. SMA is caused by mutations in the telomeric copy of the survival motor neuron 1 (SMN1) gene, but all patients retain a centromeric copy of the gene,...
Source: Current Protocols in Human Genetics - January 11, 2016 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Molecular Diagnosis of Cystic Fibrosis.
Authors: Deignan JL, Grody WW Abstract This unit describes a recommended approach to identifying causal genetic variants in an individual suspected of having cystic fibrosis. An introduction to the genetics and clinical presentation of cystic fibrosis is initially presented, followed by a description of the two main strategies used in the molecular diagnosis of cystic fibrosis: (1) an initial targeted variant panel used to detect only the most common cystic fibrosis-causing variants in the CFTR gene, and (2) sequencing of the entire coding region of the CFTR gene to detect additional rare causal CFTR varia...
Source: Current Protocols in Human Genetics - January 11, 2016 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Alternative Splicing Signatures in RNA-seq Data: Percent Spliced in (PSI).
Authors: Schafer S, Miao K, Benson CC, Heinig M, Cook SA, Hubner N Abstract Thousands of alternative exons are spliced out of messenger RNA to increase protein diversity. High-throughput sequencing of short cDNA fragments (RNA-seq) generates a genome-wide snapshot of these post-transcriptional processes. RNA-seq reads yield insights into the regulation of alternative splicing by revealing the usage of known or unknown splice sites as well as the expression level of exons. Constitutive exons are never covered by split alignments, whereas alternative exonic parts are located within highly expressed splicing ...
Source: Current Protocols in Human Genetics - October 8, 2015 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Practical Integration-Free Episomal Methods for Generating Human Induced Pluripotent Stem Cells.
Authors: Kime C, Rand TA, Ivey KN, Srivastava D, Yamanaka S, Tomoda K Abstract The advent of induced pluripotent stem (iPS) cell technology has revolutionized biomedicine and basic research by yielding cells with embryonic stem (ES) cell-like properties. The use of iPS-derived cells for cell-based therapies and modeling of human disease holds great potential. While the initial description of iPS cells involved overexpression of four transcription factors via viral vectors that integrated within genomic DNA, advances in recent years by our group and others have led to safer and higher quality iPS cells with...
Source: Current Protocols in Human Genetics - October 8, 2015 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells.
We describe in depth the latest version of our monolayer chemically defined small molecule differentiation protocol, including metabolic selection-based cardiomyocyte purification and the addition of triiodothyronine to enhance cardiomyocyte maturation. Finally, we describe a method for the dissociation of hPS cell-derived cardiomyocytes, cryopreservation, and thawing. © 2015 by John Wiley & Sons, Inc. PMID: 26439715 [PubMed - in process] (Source: Current Protocols in Human Genetics)
Source: Current Protocols in Human Genetics - October 8, 2015 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Interpreting de novo Variation in Human Disease Using denovolyzeR.
Authors: Ware JS, Samocha KE, Homsy J, Daly MJ Abstract Spontaneously arising (de novo) genetic variants are important in human disease, yet every individual carries many such variants, with a median of 1 de novo variant affecting the protein-coding portion of the genome. A recently described mutational model provides a powerful framework for the robust statistical evaluation of such coding variants, enabling the interpretation of de novo variation in human disease. Here we describe a new open-source software package, denovolyzeR, that implements this model and provides tools for the analysis of de novo co...
Source: Current Protocols in Human Genetics - October 8, 2015 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 increasingly inexpensive, and is rapidly being incorporated into clinical practice. Incidental or secondary findings, which can occur in large numbers from genomic sequencing, are a potential barrier to the utility of this new technology due to their relatively 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 h...
Source: Current Protocols in Human Genetics - October 8, 2015 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Huntington Disease: Molecular Diagnostics Approach.
Authors: Bastepe M, Xin W Abstract Huntington disease (HD) is caused by expansion of a CAG trinucleotide repeat in the first exon of the Huntingtin (HTT) gene. Molecular testing of Huntington disease for diagnostic confirmation and disease prediction requires detection of the CAG repeat expansion. There are three main types of HD genetic testing: (1) diagnostic testing to confirm or rule out disease, (2) presymptomatic testing to determine whether an at-risk individual inherited the expanded allele, and (3) prenatal testing to determine whether the fetus has inherited the expanded allele. This unit include...
Source: Current Protocols in Human Genetics - October 8, 2015 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Using the PhenX Toolkit to Add Standard Measures to a Study.
Authors: Hendershot T, Pan H, Haines J, Harlan WR, Marazita ML, McCarty CA, Ramos EM, Hamilton CM Abstract The PhenX (consensus measures for Phenotypes and eXposures) Toolkit (https://www.phenxtoolkit.org/) offers high-quality, well-established measures of phenotypes and exposures for use by the scientific community. The goal is to promote the use of standard measures, enhance data interoperability, and help investigators identify opportunities for collaborative and translational research. The Toolkit contains 395 measures drawn from 22 research domains (fields of research), along with additional collectio...
Source: Current Protocols in Human Genetics - July 5, 2015 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Glycosylation Analysis for Congenital Disorders of Glycosylation.
Authors: Li X, Raihan MA, Reynoso FJ, He M Abstract Congenital disorders of glycosylation (CDG) are a group of diseases with highly variable phenotypes and inconsistent clinical features. Since the first description of a CDG in 1980, approximately 100 disorders have been identified. Most of these are defects in protein glycosylation, although an increasing number are defects of glycolipid or proteoglycan biosynthesis. A group of biochemical markers has been used to characterize protein glycosylation abnormalities in CDG. This unit describes three protocols that can be used to measure plasma or serum carboh...
Source: Current Protocols in Human Genetics - July 5, 2015 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper.
Authors: Auler K, Broock R, Nyhan WL Abstract Hypoxanthine-guanine phosphoribosyl-transferase (HPRT) deficiency is the cause of Lesch-Nyhan disease. Adenine phosphoribosyl-transferase (APRT) deficiency causes renal calculi. The activity of each enzyme is readily determined on spots of whole blood on filter paper. This unit describes a method for detecting deficiencies of HPRT and APRT. © 2015 by John Wiley & Sons, Inc. PMID: 26132002 [PubMed - in process] (Source: Current Protocols in Human Genetics)
Source: Current Protocols in Human Genetics - July 5, 2015 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research