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Diagnosing lysosomal storage disorders: mucopolysaccharidosis type II.
Authors: Johnson BA, van Diggelen OP, Dajnoki A, Bodamer OA Abstract Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder caused by a deficiency of iduronate 2-sulfatase (IDS). Progressive, intralysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate in almost all tissues leads to multi-organ involvement in affected males but to virtual absence of symptoms in heterozygote female carriers due to preferential inactivation of the mutant allele. Diagnosis of MPS II in males is based on IDS analysis in leukocytes, fibroblasts, plasma, or dried blood spots...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Genome-scale sequencing to identify genes involved in Mendelian disorders.
Authors: Markello TC, Adams DR Abstract The analysis of genome-scale sequence data can be defined as the interrogation of a complete set of genetic instructions in a search for individual loci that produce or contribute to a pathological state. Bioinformatic analysis of sequence data requires sufficient discriminant power to find this needle in a haystack. Current approaches make choices about selectivity and specificity thresholds, and the quality, quantity, and completeness of the data in these analyses. There are many software tools available for individual, analytic component-tasks, including commercia...
Source: Current Protocols in Human Genetics - November 25, 2014 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 several 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 under particular circumstances 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 but rather the analysis and interpretation. Interpretati...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Retroviral vector production.
Authors: Miller AD Abstract In this unit, the basic protocol generates stable cell lines that produce retroviral vectors that carry selectable markers. Also included are an alternate protocol that applies when the retroviral vector does not carry a selectable marker, and another alternate protocol for rapidly generating retroviral vector preparations by transient transfection. A support protocol describes construction of the retroviral vectors. The methods for generating virus from retroviral vector plasmids rely on the use of packaging cells that synthesize all of the retroviral proteins but do not produc...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Quantitative analysis of copy number variants based on real-time LightCycler PCR.
Authors: Ma L, Chung WK Abstract Quantitative real-time PCR is PCR visualized in real time by the use of fluorescent or intercalating dyes, which are employed to measure gene expression or gene quantification including contiguous gene deletions or duplications. A simple method is described here to quantify DNA copy number from human samples. PMID: 24510682 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Human Genetics)
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Internet resources in medical genetics.
Authors: Waggoner DJ Abstract This unit presents an overview of the most commonly used Web-based information resources for clinicians seeking to apply molecular or array-based genetic testing to patient care, learn more about newborn screening, or understand the molecular basis of inherited diseases. It is also for consumers seeking advocacy or scientific/management information, and for genetics professional societies. PMID: 24510683 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Human Genetics)
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Molecular analysis of fragile X syndrome.
Authors: Basehore MJ, Friez MJ Abstract The gene responsible for Fragile X syndrome, fragile X mental retardation-1 (FMR1), contains an unstable sequence of CGG trinucleotide repeats in its promoter region. Expansions of >200 trinucleotide repeats are considered full mutations and typically lead to abnormal methylation of the region, resulting in loss of FMR1 expression. Males with loss of FMR1 protein are expected to be affected by Fragile X syndrome, while females may or may not clinically manifest features of the condition. The protocols in this unit outline the complementary use of polymerase chain ...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Nonrandom X chromosome inactivation detection.
Authors: Jones JR Abstract X chromosome inactivation patterns may be clinically useful in assessing tumor clonality, determining carrier status for certain X-linked disorders and evaluating the pathogenicity of a genetic variant identified in an X-linked gene. The protocols in this unit utilize the highly polymorphic trinucleotide repeat within the first exon of the human androgen receptor gene (AR) and the methylation-sensitive restriction enzyme HpaII to distinguish between the maternal and paternal alleles and simultaneously determine their methylation status. The data obtained from these protocols can ...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Mitochondrial genome interrogation for forensic casework and research studies.
Authors: Roby RK, Sprouse M, Phillips N, Alicea-Centeno A, Shewale S, Shore S, Paul N Abstract This unit describes methods used in the analysis of mitochondrial DNA (mtDNA) for forensic and research applications. UNIT describes procedures specifically for forensic casework where the DNA from evidentiary material is often degraded or inhibited. In this unit, protocols are described for quantification of mtDNA before amplification; amplification of the entire control region from high-quality samples as well as procedures for interrogating the whole mitochondrial genome (mtGenome); quantification of mtDNA pos...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Using VAAST to Identify Disease-Associated Variants in Next-Generation Sequencing Data.
Authors: Kennedy B, Kronenberg Z, Hu H, Moore B, Flygare S, Reese MG, Jorde LB, Yandell M, Huff C Abstract The VAAST pipeline is specifically designed to identify disease-associated alleles in next-generation sequencing data. In the protocols presented in this paper, we outline the best practices for variant prioritization using VAAST. Examples and test data are provided for case-control, small pedigree, and large pedigree analyses. These protocols will teach users the fundamentals of VAAST, VAAST 2.0, and pVAAST analyses. Curr. Protoc. Hum. Genet. 81:6.14.1-6.14.25. © 2014 by John Wiley & Sons, Inc. ...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Using XHMM Software to Detect Copy Number Variation in Whole-Exome Sequencing Data.
Authors: Fromer M, Purcell SM Abstract Copy number variation (CNV) has emerged as an important genetic component in human diseases, which are increasingly being studied for large numbers of samples by sequencing the coding regions of the genome, i.e., exome sequencing. Nonetheless, detecting this variation from such targeted sequencing data is a difficult task, involving sorting out signal from noise, for which we have recently developed a set of statistical and computational tools called XHMM. In this unit, we give detailed instructions on how to run XHMM and how to use the resulting CNV calls in biologic...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Genetic tests: clinical validity and clinical utility.
Authors: Burke W Abstract When evaluating the appropriate use of new genetic tests, clinicians and health care policymakers must consider the accuracy with which a test identifies a patient's clinical status (clinical validity) and the risks and benefits resulting from test use (clinical utility). Genetic tests in current use vary in accuracy and potential to improve health outcomes, and these test properties may be influenced by testing technology and the clinical setting in which the test is used. This unit defines clinical validity and clinical utility, provides examples, and considers the implications ...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Design of large-insert jumping libraries for structural variant detection using illumina sequencing.
Authors: Hanscom C, Talkowski M Abstract Next-generation sequencing is an important and efficient tool for the identification of structural variation, particularly balanced chromosomal rearrangements, because such events are not routinely detected by microarray and localization of altered regions by karyotype is imprecise. Indeed, the degree of resolution that can be obtained through next-generation technologies enables elucidation of precise breakpoints and has facilitated the discovery of numerous pathogenic loci in human disease and congenital anomalies. The protocol described here explains one type of ...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

ENU Mutagenesis in the Mouse.
Authors: Stottmann R, Beier D Abstract This unit describes the treatment of laboratory mice with the mutagen N-ethyl-N-nitrosourea (ENU) to induce very highly increased rates of mutation throughout the genome. Further, it describes several popular mating schemes designed to produce animals displaying phenotypes associated with the induced mutations. Curr. Protoc. Hum. Genet. 82:15.4.1-15.4.10 © 2014 by John Wiley & Sons, Inc. PMID: 25042716 [PubMed - in process] (Source: Current Protocols in Human Genetics)
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research

Diagnosis of lysosomal storage disorders: Gaucher disease.
Authors: Johnson BA, Dajnoki A, Bodamer O Abstract Gaucher Disease (GD) is a progressive lysosomal storage disorder caused by deficiency of glucocerebrosidase (GBA). The clinical phenotype follows a spectrum ranging from severe early-onset to milder late-onset disease. The absence of neurological involvement defines GD type I, whereas neuronopathic features define GD type II and III. Early diagnosis may be important for timely initiation of enzyme replacement therapy to prevent disease complications, although the enzyme does not cross the blood brain barrier. Diagnosis of GD can be readily achieved by anal...
Source: Current Protocols in Human Genetics - November 25, 2014 Category: Genetics & Stem Cells Tags: Curr Protoc Hum Genet Source Type: research