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Current Protocols in Human Genetics

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https://www.readbyqxmd.com/read/28075488/generating-exome-enriched-sequencing-libraries-from-formalin-fixed-paraffin-embedded-tissue-dna-for-next-generation-sequencing
#1
Beth A Marosy, Brian D Craig, Kurt N Hetrick, P Dane Witmer, Hua Ling, Sean M Griffith, Benjamin Myers, Elaine A Ostrander, Janet L Stanford, Lawrence C Brody, Kimberly F Doheny
This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform...
January 11, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/28075487/molecular-analysis-of-gene-rearrangements-and-mutations-in-acute-leukemias-and-myeloid-neoplasms
#2
Lynette M Sholl, Janina Longtine, Frank C Kuo
A subset of acute leukemias and other myeloid neoplasms contains specific genetic alterations, many of which are associated with unique clinical and pathologic features. These alterations include chromosomal rearrangements leading to oncogenic fusion proteins or alteration of gene expression by juxtaposing oncogenes to enhancer elements, as well as mutations leading to aberrant activation of a variety of proteins critical to hematopoietic progenitor cell proliferation and differentiation. Molecular analysis is central to diagnosis and clinical management of leukemias, permitting genetic confirmation of a clinical and histologic impression, providing prognostic and predictive information, and facilitating detection of minimal residual disease...
January 11, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/28075486/highly-expandable-human-ips-cell-derived-neural-progenitor-cells-npc-and-neurons-for-central-nervous-system-disease-modeling-and-high-throughput-screening
#3
Chialin Cheng, Daniel M Fass, Kat Folz-Donahue, Marcy E MacDonald, Stephen J Haggarty
Reprogramming of human somatic cells into induced pluripotent stem (iPS) cells has greatly expanded the set of research tools available to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) disorders. Realizing the promise of iPS cell technology for the identification of novel therapeutic targets and for high-throughput drug screening requires implementation of methods for the large-scale production of defined CNS cell types. Here we describe a protocol for generating stable, highly expandable, iPS cell-derived CNS neural progenitor cells (NPC) using multi-dimensional fluorescence activated cell sorting (FACS) to purify NPC defined by cell surface markers...
January 11, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/28075485/culturing-and-neuronal-differentiation-of-human-dental-pulp-stem-cells
#4
Sarita Goorha, Lawrence T Reiter
A major issue in studying human neurogenetic disorders, especially rare syndromes affecting the nervous system, is the ability to grow neuronal cultures that accurately represent these disorders for analysis. Although there has been some success in generating induced pluripotent stem (iPS) cells from both skin and blood, there are still limitations to the collection and production of iPS cells from these biospecimens. We have had significant success in collecting and growing human dental pulp stem (DPS) cells from exfoliated teeth sent to our laboratory by the parents of children with a variety of rare neurogenetic syndromes...
January 11, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/28075484/assay-for-transposase-accessible-chromatin-using-sequencing-atac-seq-data-analysis
#5
Kristy L S Miskimen, E Ricky Chan, Jonathan L Haines
The study of epigenetic properties of the human genome, including structural modifications of DNA and chromatin, has increased tremendously as mounting evidence has demonstrated how much epigenetics affects human gene expression. Buenrostro et al. have developed a rapid method, requiring low numbers of living cells as input, for examining chromatin accessibility across the epigenome, known as the assay for transposase-accessible chromatin using sequencing (ATAC-seq). The overall goal of this unit is to provide a thorough ATAC-seq data analysis plan, as well as describe how primary human blood samples can be processed for use in ATAC-seq...
January 11, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/28075483/detecting-apc-gene-mutations-in-familial-adenomatous-polyposis-fap
#6
Babi Ramesh Reddy Nallamilli, Madhuri Hegde
Hereditary forms of colorectal cancer (CRC) account for up to 5% of total cases. Familial adenomatous polyposis (FAP) is an autosomal dominant condition affecting nearly 1 in 5000 people and accounts for only about 1% of all CRCs. It is characterized by the progressive development of hundreds to thousands of adenomatous colon polyps. The gene associated with FAP (APC) contains 15 coding exons. The mutation spectrum of the APC gene is broad in that 87% of causative mutations are point mutations (including other sequence variants) and around 10% to 15% are intragenic deletions and duplications...
January 11, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/28075482/human-induced-pluripotent-stem-hips-cells-from-urine-samples-a-non-integrative-and-feeder-free-reprogramming-strategy
#7
Clara Steichen, Karim Si-Tayeb, Fanny Wulkan, Thayane Crestani, Graça Rosas, Rafael Dariolli, Alexandre C Pereira, Jose E Krieger
Human induced pluripotent stem (hiPS) cell technology has already revolutionized some aspects of fundamental and applied research such as study of disease mechanisms and pharmacology screening. The first clinical trial using hiPS cell-derived cells began in Japan, only 10 years after the publication of the proof-of concept article. In this exciting context, strategies to generate hiPS cells have evolved quickly, tending towards non-invasive protocols to sample somatic cells combined with "safer" reprogramming strategies...
January 11, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/28075481/diagnosis-of-spinocerebellar-ataxias-caused-by-trinucleotide-repeat-expansions
#8
Joanne E Martindale
Spinocerebellar ataxias (SCAs) are a group of disorders that are both clinically and genetically heterogeneous. They usually demonstrate onset in adulthood, but some forms may have juvenile or infantile onset. There are many different types of SCA, demonstrating different modes of inheritance and types of mutation. The most common forms are due to dominantly inherited expansions in trinucleotide repeat sequences located within the coding region of the relevant genes, and these are readily identifiable by molecular genetic testing...
January 11, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27727440/genetic-risk-scores
#9
Jessica N Cooke Bailey, Robert P Igo
The generation of genome-wide variation data has 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 disease susceptibility is to aggregate the risk effects of these loci into a single genetic risk score...
October 11, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27727439/analysis-of-heritability-using-genome-wide-data
#10
Jacob B Hall, William S Bush
Most analyses of genome-wide association data consider each variant independently without considering or adjusting for the genetic background present in the rest of the genome. New approaches to genome analysis use representations of genomic sharing to better account for confounding factors like population stratification or to directly approximate heritability through the estimated sharing of individuals in a dataset. These approaches use mixed linear models, which relate genotypic sharing to phenotypic sharing, and rely on the efficient computation of genetic sharing among individuals in a dataset...
October 11, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27727438/cosmic-high-resolution-cancer-genetics-using-the-catalogue-of-somatic-mutations-in-cancer
#11
S A Forbes, D Beare, N Bindal, S Bamford, S Ward, C G Cole, M Jia, C Kok, H Boutselakis, T De, Z Sondka, L Ponting, R Stefancsik, B Harsha, J Tate, E Dawson, S Thompson, H Jubb, P J Campbell
COSMIC (http://cancer.sanger.ac.uk) is an expert-curated database of somatic mutations in human cancer. Broad and comprehensive in scope, recent releases in 2016 describe over 4 million coding mutations across all human cancer disease types. Mutations are annotated across the entire genome, but expert curation is focused on over 400 key cancer genes. Now encompassing the majority of molecular mutation mechanisms in oncogenetics, COSMIC additionally describes 10 million non-coding mutations, 1 million copy-number aberrations, 9 million gene-expression variants, and almost 8 million differentially methylated CpGs...
October 11, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27727437/molecular-diagnosis-of-myotonic-dystrophy
#12
Sujata Chakraborty, Matteo Vatta, Linda L Bachinski, Ralf Krahe, Stephen Dlouhy, Shaochun Bai
Myotonic dystrophy types 1 (DM1) and 2 (DM2) are autosomal dominant, microsatellite repeat expansion disorders that affect muscle function. Myotonic dystrophy type 1 is caused by CTG repeat expansion in the 3' UTR region of the DMPK gene. Patients with DM2 have expansion of CCTG repeats in intron 1 of the CNBP gene. In this unit, we review and discuss the clinical phenotypes, genetic mutations causing the diseases, and the molecular diagnostic approaches and tools that are used to determine repeat sizes in DM1/2...
October 11, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27727436/acylglycine-analysis-by-ultra-performance-liquid-chromatography-tandem-mass-spectrometry-uplc-ms-ms
#13
Judith A Hobert, Aiping Liu, Marzia Pasquali
Quantitative analysis of urine acylglycines has shown to be a highly sensitive and specific method with proven clinical utility for the diagnosis of several inherited metabolic disorders including: medium chain acyl-CoA dehydrogenase deficiency, multiple acyl-CoA dehydrogenase deficiency, short chain acyl-CoA dehydrogenase deficiency, 3-methylcrotonyl-CoA carboxylase deficiency, 2-methylbutyryl-CoA dehydrogenase deficiency, isovaleric acidemia, propionic academia, and isobutyryl-CoA dehydrogenase deficiency...
October 11, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27727435/pronuclear-injection-based-targeted-transgenesis
#14
Samantha L P Schilit, Masato Ohtsuka, Rolen M Quadros, Channabasavaiah B Gurumurthy
Microinjection of DNA expression cassettes into fertilized zygotes has been a standard method for generating transgenic animal models. While efficient, the injected DNA integrates randomly into the genome, leading to potential disruption of endogenous genes or regulatory elements, variation in copy number, or integration into heterochromatic regions that inhibit transgene expression. A recently developed method addresses such pitfalls of traditional transgenesis by targeting the transgene to predetermined sites in the genome that can safely harbor exogenous DNA...
October 11, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27727434/high-risk-screening-of-fabry-disease-analysis-of-fifteen-urinary-methylated-and-non-methylated-gb3-isoforms-using-tandem-mass-spectrometry
#15
Mona Abaoui, Michel Boutin, Pamela Lavoie, Christiane Auray-Blais
Fabry disease is a multisystemic, X-linked lysosomal storage disorder caused by mutations in the GLA gene, leading to α-galactosidase A deficiency and resulting in the accumulation of glycosphingolipids in different tissues and biological fluids. Glycosphingolipid biomarkers, such as globotriaosylceramide (Gb3 ) isoforms, globotriaosylsphingosine (lyso-Gb3 ) and related analogs, and galabiosylceramide (Ga2 ) isoforms and analogs, are found to be abnormally increased in urine and in plasma of Fabry patients and have the potential to be used as specific biomarkers of the disease...
October 11, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27367164/quality-control-for-the-illumina-humanexome-beadchip
#16
Robert P Igo, Jessica N Cooke Bailey, Jane Romm, Jonathan L Haines, Janey L Wiggs
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...
July 1, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27037490/reporting-of-diagnostic-cytogenetic-results
#17
Anne B S Giersch, Frederick R Bieber, Adrian M Dubuc, Jonathan A Fletcher, Azra H Ligon, Heather Mason-Suares, Cynthia C Morton, Stanislawa Weremowicz, Sheng Xiao, Paola Dal Cin
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 type (GTG-banding, FISH, or chromosomal microarray)...
April 1, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27037489/using-clinvar-as-a-resource-to-support-variant-interpretation
#18
Steven M Harrison, Erin R Riggs, Donna R Maglott, Jennifer M Lee, Danielle R Azzariti, Annie Niehaus, Erin M Ramos, Christa L Martin, Melissa J Landrum, Heidi L Rehm
ClinVar is a freely accessible, public archive of reports of the relationships among genomic variants and phenotypes. To facilitate evaluation of the clinical significance of each variant, ClinVar aggregates submissions of the same variant, displays supporting data from each submission, and determines if the submitted clinical interpretations are conflicting or concordant. The unit describes how to (1) identify sequence and structural variants of interest in ClinVar by multiple searching approaches, including Variation Viewer and (2) understand the display of submissions to ClinVar and the evidence supporting each interpretation...
April 1, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27037488/overview-of-clinical-cytogenetics
#19
Patrick R Gonzales, Andrew J Carroll, Bruce R Korf
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...
April 1, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27037487/quantitative-analysis-of-total-plasma-homocysteine-by-lc-ms-ms
#20
Libin Yuan, J Daniel Sharer
Homocysteine is a nonessential, sulfur-containing amino acid involved in one-carbon (folate) metabolism. A number of inherited and acquired conditions cause increased accumulation of this metabolite in blood (homocysteinemia) and other biofluids. Homocysteinemia is a risk factor for cardiovascular disease, including recurrent thrombosis. Accurate measurement of total plasma homocysteine is an important element in the diagnostic evaluation of these disorders. While a number of different methods have been developed for this purpose, the focus of this unit will be on a specific technique utilizing liquid chromatography-tandem mass spectrometry, which provides several advantages in terms of speed, sensitivity, and specificity...
April 1, 2016: Current Protocols in Human Genetics
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