Genetic Risk Scores.
Authors: Igo RP, Kinzy TG, Cooke Bailey JN Abstract Genome-wide variation data with millions of genetic markers have become commonplace. However, the potential for interpretation and application of these data for clinical assessment of outcomes of interest, and prediction of disease risk, is currently not fully realized. Many common complex diseases now have numerous, well-established risk loci and likely harbor many genetic determinants with effects too small to be detected at genome-wide levels of statistical significance. A simple and intuitive approach for converting genetic data to a predictive measure of dise...
Source: Current Protocols in Human Genetics - November 26, 2019 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Approaches to Whole Mitochondrial Genome Sequencing on the Oxford Nanopore MinION.
This article describes three approaches to extract, prepare, and sequence mitochondrial DNA on the Oxford Nanopore MinION device. Two of the workflows include enrichment of mitochondrial DNA prior to sequencing, whereas the other relies on direct sequencing of native genomic DNA to allow for simultaneous assessment of the nuclear and mitochondrial genomes. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Enrichment-free mitochondrial DNA sequencing Alternate Protocol 1: Mitochondrial DNA sequencing following enrichment with polymerase chain reaction (PCR) Alternate Protocol 2: Mitochondrial DNA sequencing followi...
Source: Current Protocols in Human Genetics - November 20, 2019 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Overview of Specifications to the ACMG/AMP Variant Interpretation Guidelines.
This article summarizes the approaches to, and rationale for, specifying three evidence categories (population frequency data, variant type and location, and case-level data), including available resources and a quantitative framework that can inform the specification process. © 2019 by John Wiley & Sons, Inc. PMID: 31479589 [PubMed - in process] (Source: Current Protocols in Human Genetics)
Source: Current Protocols in Human Genetics - September 5, 2019 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Encoding Clinical Data with the Human Phenotype Ontology for Computational Differential Diagnostics.
This article explains how to choose an optimal set of HPO terms for these cases and enter them with software, such as PhenoTips and PatientArchive, and demonstrates how to use Phenomizer and Exomiser to generate a computational differential diagnosis. © 2019 by John Wiley & Sons, Inc. PMID: 31479590 [PubMed - in process] (Source: Current Protocols in Human Genetics)
Source: Current Protocols in Human Genetics - September 5, 2019 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Best Practices for Illumina Library Preparation.
Authors: Bronner IF, Quail MA Abstract In this unit, we describe a set of protocols and recommendations for Illumina library preparation. We review best practices in template quantitation methods; template fragmentation methodologies; solid-phase reverse-immobilization cleanup, including buffer exchange and size selection; end repair, A-tailing, and adapter ligation; indexing strategies; considerations regarding whether to use polymerase chain reaction; final library quantification methodologies; and normalization and pooling strategies. These workflows are applicable to both high-throughput and low-throughput Illu...
Source: Current Protocols in Human Genetics - June 21, 2019 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Analytical Methods for Quantitative Plasma Carnitine Determination.
Authors: Zimmerman CN, Sharer JD Abstract Carnitine is an essential molecule for mitochondrial beta-oxidation of long-chain fatty acids and other cellular functions. Several rare, inherited disorders of carnitine metabolism occur in humans, and secondary carnitine deficiency is an important feature in a variety of clinical settings. Many of these conditions can be detected via quantitative analysis of free and esterified carnitine in plasma or urine, which thus offers an effective means for assessing the transport and initial processing of fatty acids. Here, we describe some of the methods most commonly employed fo...
Source: Current Protocols in Human Genetics - June 21, 2019 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Genotype Imputation in Genome-Wide Association Studies.
This article reviews the general concepts behind imputation, describes imputation approaches and methods for various types of genotype data, including family-based data, and identifies web-based resources that can be used in different steps of the imputation process. For practical application, it provides a step-by-step guide to implementation of a two-step imputation process consisting of phasing of the study genotypes and the imputation of reference panel genotypes into the study haplotypes. In addition, this review describes recently developed haplotype reference panel resources and online imputation servers that are ca...
Source: Current Protocols in Human Genetics - June 21, 2019 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Methods for the Analysis and Interpretation for Rare Variants Associated with Complex Traits.
Authors: Weissenkampen JD, Jiang Y, Eckert S, Jiang B, Li B, Liu DJ Abstract With the advent of Next Generation Sequencing (NGS) technologies, whole genome and whole exome DNA sequencing has become affordable for routine genetic studies. Coupled with improved genotyping arrays and genotype imputation methodologies, it is increasingly feasible to obtain rare genetic variant information in large datasets. Such datasets allow researchers to gain a more complete understanding of the genetic architecture of complex traits caused by rare variants. State-of-the-art statistical methods for the statistical genetics analysis...
Source: Current Protocols in Human Genetics - March 10, 2019 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Conducting a Reproducible Mendelian Randomization Analysis Using the R Analytic Statistical Environment.
Authors: Rasooly D, Patel CJ Abstract Mendelian randomization (MR) is defined as the utilization of genetic variants as instrumental variables to assess the causal relationship between an exposure and an outcome. By leveraging genetic polymorphisms as proxy for an exposure, the causal effect of an exposure on an outcome can be assessed while addressing susceptibility to biases prone to conventional observational studies, including confounding and reverse causation, where the outcome causes the exposure. Analogous to a randomized controlled trial where patients are randomly assigned to subgroups based on different t...
Source: Current Protocols in Human Genetics - January 17, 2019 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Simultaneous Targeted Methylation Sequencing (sTM-Seq).
Authors: Asmus N, Papale LA, Madrid A, Alisch RS Abstract Mapping patterns of DNA methylation throughout the epigenome are critical to our understanding of several important biological and regulatory functions, such as transcriptional regulation, genomic imprinting, and embryonic development. The development and rapid advancement of next-generation sequencing (NGS) technologies have provided clinicians and researchers with accurate and reliable read-outs of genomic and epigenomic information at the nucleotide level. Such improvements have significantly lowered the cost required for genome-wide sequencing, facilitat...
Source: Current Protocols in Human Genetics - January 10, 2019 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Using Electronic Health Records To Generate Phenotypes For Research.
We describe here common and emerging electronic phenotyping approaches applied to electronic health records, as well as current limitations of both the approaches and the biases associated with these clinically collected data that impact their use in research. © 2018 by John Wiley & Sons, Inc. PMID: 30516347 [PubMed - as supplied by publisher] (Source: Current Protocols in Human Genetics)
Source: Current Protocols in Human Genetics - December 6, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Strategies for Pathway Analysis Using GWAS and WGS Data.
Authors: White MJ, Yaspan BL, Veatch OJ, Goddard P, Risse-Adams OS, Contreras MG Abstract Single-allele study designs, commonly used in genome-wide association studies (GWAS) as well as the more recently developed whole genome sequencing (WGS) studies, are a standard approach for investigating the relationship of common variation within the human genome to a given phenotype of interest. However, single-allele association results published for many GWAS studies represent only the tip of the iceberg for the information that can be extracted from these datasets. The primary analysis strategy for GWAS entails associati...
Source: Current Protocols in Human Genetics - November 4, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Glucose-Responsiveness of Pancreatic β-Like (GRP β-L) Cells Generated from Human Pluripotent Stem Cells.
Glucose-Responsiveness of Pancreatic β-Like (GRP β-L) Cells Generated from Human Pluripotent Stem Cells. Curr Protoc Hum Genet. 2018 Oct 18;:e71 Authors: Rajaei B, Massumi M, Wheeler M Abstract The International Diabetic Federation estimated that 415 million adults currently have diabetes and 318 million adults had impaired glucose tolerance, putting them at high risk of developing diabetes in the future. In Type 1 Diabetes (T1D), the β cells are lost because of autoimmune reactions. Although islet transplantation has been a promising therapy for T1D, it is greatly limited by pancreatic ...
Source: Current Protocols in Human Genetics - October 20, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Obtaining a Genetic Family History Using Computer-Based Tools.
Authors: Li W, Murray MF, Giovanni MA Abstract Family health history has long been known to be a powerful predictor of individual disease risk. It can be obtained prior to DNA sequencing in order to examine inheritance patterns, to be used as a proxy for genetic information, or as a tool to guide decision-making on the utility of diagnostic genetic testing. Increasingly, it is also being obtained retrospectively from sequenced individuals to examine familial disease penetrance and to identify at-risk relatives for cascade testing. The collection of adequate family history information to screen patients for disease ...
Source: Current Protocols in Human Genetics - October 20, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Quantification of Muscle Contraction In Vitro and In Vivo Using MUSCLEMOTION Software: From Stem Cell-Derived Cardiomyocytes to Zebrafish and Human Hearts.
We describe high-speed and disturbance-free acquisition of images from either electrically paced or non-paced human pluripotent stem cell-derived cardiomyocytes, isolated adult cardiomyocytes, zebrafish hearts, and human echocardiograms. Recordings are then used as input for automated batch analysis by the MUSCLEMOTION software tool configured with specific settings and parameters tailored to the recording technique. Details on accuracy, interpretation, and troubleshooting are discussed. Acquisition duration depends on the experimental setup and aim, but quantification of drug or disease responses in an in vitro muscle mod...
Source: Current Protocols in Human Genetics - September 28, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

MicroRNA Isolation from Plasma for Real-Time qPCR Array.
Authors: Witvrouwen I, Gevaert AB, Van Craenenbroeck EM, Van Craenenbroeck AH Abstract MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level by mRNA degradation or suppression of translation. Their stability in plasma makes them attractive biomarkers. Since many plasma microRNA isolation procedures exist and the yield can be highly variable, we recently optimized the microRNA isolation and preamplification procedure using the mirVana PARIS kit (Thermo Fisher Scientific) for miRNA quantification with TaqMan Low Density Arrays in plasma samples. The method here is slightl...
Source: Current Protocols in Human Genetics - September 16, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Multicolor Fluorescence In Situ Hybridization (FISH) Approaches for Simultaneous Analysis of the Entire Human Genome.
Authors: Zhang C, Cerveira E, Rens W, Yang F, Lee C Abstract Analysis of the organization of the human genome is vital for understanding genetic diversity, human evolution, and disease pathogenesis. A number of approaches, such as multicolor fluorescence in situ hybridization (FISH) assays, cytogenomic microarray (CMA), and next-generation sequencing (NGS) technologies, are available for simultaneous analysis of the entire human genome. Multicolor FISH-based spectral karyotyping (SKY), multiplex FISH (M-FISH), and Rx-FISH may provide rapid identification of interchromosomal and intrachromosomal rearrangements as we...
Source: Current Protocols in Human Genetics - September 16, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

A Guided Protocol for Array Based T2C: A High-Quality Selective High-Resolution High-Throughput Chromosome Interaction Capture.
Authors: Knoch TA Abstract After now more than 170 years of research the dynamic three-dimensional chromatin architecture of genomes and the co-evolved interaction networks of regulatory elements which create genome function - i.e. the storage, expression, and finally replication of genetic information - involves ever more investigative efforts in respect to not only the pure understanding of living organisms, but also diagnosis, treatment, and even future genome engineering. To study genomic interactions, we developed a novel and superior high-quality selective high-resolution, high-throughput chromosome inte...
Source: Current Protocols in Human Genetics - September 12, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Pancreatic Beta Cell Differentiation From Human Pluripotent Stem Cells.
Authors: Sui L, Leibel RL, Egli D Abstract Insulin-expressing beta cells are crucial for the maintenance of systemic glucose homeostasis. Elucidation of the molecular and cellular mechanisms of beta cell development, expansion, survival, and function are required for full understanding of the molecular pathogenesis of diabetes. However, access to human beta cells for such studies is limited by virtue of the logistics of acquisition, prior medical status of donor, and imperfect culture systems for maintaining beta cell identity and function after isolation from human pancreas. Here, a technique for generation of bet...
Source: Current Protocols in Human Genetics - September 9, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

In vivo and In vitro methods to identify DNA sequence variants that alter RNA Splicing.
Authors: Patel PN, Gorham JM, Ito K, Seidman CE Abstract Identification of sequence variants that create or eliminate splice sites has proven to be a significant challenge and represents one of many roadblocks in the clinical interpretation of rare genetic variation. Current methods of identifying splice altering sequence variants exist, however, these are limited by an imperfect understanding of splice signals and cumbersome functional assays. We have recently developed a computational tool that prioritizes putative splice-altering sequence variants, and a moderate-throughput minigene assay that confirms the varia...
Source: Current Protocols in Human Genetics - July 25, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Computational Prediction of Position Effects of Human Chromosome Rearrangements.
Authors: Zepeda-Mendoza CJ, Menon S, Morton CC Abstract Balanced and apparently balanced chromosome abnormalities (BCAs) have long been known to generate disease through position effects, either by altering local networks of gene regulation or positioning genes in architecturally different chromosome domains. Despite these observations, identification of distally affected genes by BCAs is oftentimes neglected, especially when predicted gene disruptions are found elsewhere in the genome. In this unit, we provide detailed instructions on how to run a computational pipeline that identifies relevant candidates of non-c...
Source: Current Protocols in Human Genetics - July 25, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

PLINK: Key Functions for Data Analysis.
Authors: Slifer SH Abstract Genetic data analysis of large numbers of single nucleotide variants (SNVs), including genome-wide association studies (GWAS), exome chips, and whole exome (WES) or whole-genome (WGS) sequencing data, requires well defined processing steps. As a result, several freely available analytic toolkits have been developed to streamline these processes. Among these, PLINK is the most comprehensive in terms of its quality control and analytic modules, although its focus remains on SNVs. PLINK fulfills two analytic needs-aiding the process of performing quality control (QC) on large data sets and ...
Source: Current Protocols in Human Genetics - July 25, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Droplet Digital PCR with EvaGreen Assay: Confirmational Analysis of Structural Variants.
Authors: Tai AC, Parfenov M, Gorham JM Abstract DNA structural variants can be analyzed by droplet digital PCR (ddPCR), a water-oil microfluidics and fluorescence technology to quantify target nucleic acids with extreme precision and sensitivity. Traditional ddPCR uses expensive fluorescent oligonucleotide probes that require extensive optimization. Here we describe a variation of ddPCR using a DNA-binding dye (EvaGreen), whose properties allow target products to be effectively quantified at a significantly lower cost. © 2018 by John Wiley & Sons, Inc. PMID: 30040209 [PubMed - in process] (Source: Curr...
Source: Current Protocols in Human Genetics - July 25, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Pedigree Selection and Information Content.
Authors: Vardarajan BN, Beecham GW, Haines JL Abstract In this article, we discuss strategies for selection of families and family members for genetic studies. We will evaluate strategies to sample large families with multiply affected members, sibships, and nuclear families. In addition, we have added a section to discuss sub-sampling within pedigrees for large sequencing studies, particularly when genome-wide SNP chips are available on all members of a pedigree. The type of family sampled for a study will determine the statistical analyses and power of discovery of genetic findings. We will evaluate study designs...
Source: Current Protocols in Human Genetics - July 25, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Assessing Skewed X-Chromosome Inactivation.
We describe a simple and straightforward method for detection and characterization of X-chromosome inactivation in females and/or individuals with more than one X chromosome. The X-chromosome inactivation pattern is visualized on a single-cell level using 5-ethynyl-2-deoxyuridine (EdU) instead of the previously widely applied 5-bromo-2'-deoxyuridine (BUdR). The fluorochrome-labeled nucleoside analog EdU is incorporated into late-replication chromosomal regions of living blood cells in vitro; thus, it can also be used to specifically highlight the inactive X chromosome within a cytogenetic preparation. The EdU-based test fo...
Source: Current Protocols in Human Genetics - July 11, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Capture Hi-C Library Generation and Analysis to Detect Chromatin Interactions.
Authors: Orlando G, Kinnersley B, Houlston RS Abstract Chromosome conformation capture (3C), coupled with next-generation sequencing (Hi-C), provides a means for deciphering not only the principles underlying genome folding and architecture, but more broadly, the role 3D chromatin structure plays in gene regulation and the replication and repair of DNA. The recently implemented modification, in situ Hi-C, maintains nuclear integrity during digestion and ligation steps, reducing random ligation of Hi-C fragments. Although Hi-C allows for genome-wide characterization of chromatin contacts, it requires high-depth sequ...
Source: Current Protocols in Human Genetics - July 8, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Efficient Differentiation of Human Pluripotent Stem Cells to Endothelial Cells.
Authors: Gu M Abstract Endothelial cells (ECs) line the interior surface of blood and lymphatic vessels, and play a key role in a variety of physiological or pathological processes such as thrombosis, inflammation, or vascular wall remodeling. Human-induced pluripotent stem cell (iPSCs)-derived ECs provide a new opportunity for vascular regeneration and serve as a model to study the mechanism and to screen for novel therapies. We use developmental cues in a monolayer differentiation approach to efficiently generate mesoderm cells from iPSCs via small-molecule activation of WNT signaling in chemically defined medium...
Source: Current Protocols in Human Genetics - July 8, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Reporting of Clinical Genome Sequencing Results.
Authors: Song C, Duzkale H, Shen J Abstract High-throughput sequencing and high-performance computing technologies have become powerful tools in clinical genetic diagnosis of hereditary disorders and genetic screening of healthy individuals to provide information for the diagnosis, treatment, and prevention of diseases or impairment and assessment of health. For patients with undiagnosed disorders, including many rare disorders, the whole-genome sequencing (WGS) test may end the diagnostic odyssey, ultimately guiding clinical care for them and their families. A clinical WGS test relies on high-quality genome-sequen...
Source: Current Protocols in Human Genetics - July 8, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Using XCAVATOR and EXCAVATOR2 to Identify CNVs from WGS, WES, and TS Data.
Authors: D'Aurizio R, Semeraro R, Magi A Abstract Copy Number Variants (CNVs) are structural rearrangements contributing to phenotypic variation but also associated with many disease states. In recent years, the identification of CNVs from high-throughput sequencing experiments has become a common practice for both research and clinical purposes. Several computational methods have been developed so far. In this unit, we describe and give instructions on how to run two read count-based tools, XCAVATOR and EXCAVATOR2, which are tailored for the detection of both germline and somatic CNVs from different sequencing exp...
Source: Current Protocols in Human Genetics - July 7, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Preparation, Culture, and Analysis of Amniotic Fluid Samples.
Authors: Miron PM Abstract Amniotic fluid obtained via amniocentesis provides a source of fetal material used in prenatal diagnosis. The fluid may be used directly for biochemical analyses, fluorescence in situ hybridization (FISH), and isolation of DNA for molecular studies, including chromosomal microarray analysis (CMA). The fluid is typically cultured as a source of metaphase cells for chromosome analysis and to provide additional material for biochemical and DNA-based testing. This unit describes an in situ method for the preparation, culture, and harvest of amniotic fluid samples for metaphase chromosome anal...
Source: Current Protocols in Human Genetics - June 30, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

CRISPR/Cas9-Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells.
Authors: Sharma A, Toepfer CN, Ward T, Wasson L, Agarwal R, Conner DA, Hu JH, Seidman CE Abstract Human induced pluripotent stem cells (hiPSCs) can be used to mass produce surrogates of human tissues, enabling new advances in drug screening, disease modeling, and cell therapy. Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology use homology-directed repair (HDR) to efficiently generate custom hiPSC lines harboring a variety of genomic insertions and deletions. Thus, hiPSCs that encode an endogenous protein fused to a fluorescent reporter protein c...
Source: Current Protocols in Human Genetics - January 25, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Balanced Chromosomal Rearrangement Detection by Low-Pass Whole-Genome Sequencing.
Authors: Dong Z, Ye L, Yang Z, Chen H, Yuan J, Wang H, Guo X, Li Y, Wang J, Chen F, Cheung SW, Morton CC, Jiang H, Choy KW Abstract Balanced chromosomal rearrangements (or balanced chromosome abnormalities, BCAs) are common chromosomal structural variants. Emerging studies have demonstrated the feasibility of using whole-genome sequencing (WGS) for detection of BCA-associated breakpoints, but the requirement for a priori knowledge of the rearranged regions from G-banded chromosome analysis limits its application. The protocols described here are based on low-pass WGS for detecting BCA events independent from chromo...
Source: Current Protocols in Human Genetics - January 25, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Biosafety in Handling Gene Transfer Vectors.
Authors: Swindle S Abstract This unit is devoted to safety issues that must be considered when generating and working with the most common vectors under development for human gene therapy today. © 2018 by John Wiley & Sons, Inc. PMID: 29364521 [PubMed - in process] (Source: Current Protocols in Human Genetics)
Source: Current Protocols in Human Genetics - January 25, 2018 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

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, small mol...
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-differentiatio...
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 discussion o...
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. Interpretation o...
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 described are ...
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 differentiate homotyp...
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 providing CNV de...
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 not well s...
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 Protocols in Human Genetics)
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 Protocols in Human Genetics)
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. Lynch synd...
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 inferrin...
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 this prot...
Source: Current Protocols in Human Genetics - April 8, 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 as a modi...
Source: Current Protocols in Human Genetics - April 8, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Molecular Analysis of Genetic Markers for Non-Hodgkin Lymphomas.
Authors: Sholl LM, Longtine J, Kuo FC Abstract Molecular analysis complements the clinical and histopathologic tools used to diagnose and subclassify hematologic malignancies. The presence of clonal antigen-receptor gene rearrangements can help to confirm the diagnosis of a B or T cell lymphoma and can serve as a fingerprint of that neoplasm to be used in identifying concurrent disease at disparate sites or recurrence at future time points. Certain lymphoid malignancies harbor a characteristic chromosomal translocation, a finding that may have significant implications for an individual's prognosis or response to th...
Source: Current Protocols in Human Genetics - April 8, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Overview of Genetic Diagnosis in Cancer.
Authors: Korf BR, Mikhail FM Abstract Both cytogenetic and molecular genetic studies can contribute to the management of patients with cancer. In some cases, genetic markers are specific to particular tumor types and are useful in diagnosis. This can be helpful in distinguishing histologically similar tumors that may respond differently to treatment and can sometimes be of prognostic value. Genetic markers can also be tools for following the response of a tumor to therapy, providing a sensitive means to detect relapse. This introductory unit considers some of the types of genetic changes that occur in association w...
Source: Current Protocols in Human Genetics - April 8, 2017 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research