Validation and implementation of a custom 21-gene panel next-generation sequencing assay for myeloid neoplasms

Rapid and reliable mutational analysis of myeloid neoplasms is increasingly important for diagnostic, prognostic and therapeutic reasons. In this article we describe the development and validation of a custom next-generation sequencing (NGS) assay that reliably tests across a broad range of myeloid neoplasms, including AML, MDS, and myeloproliferative neoplasms. The lllumina TruSeq Custom Amplicon panel was designed to detect variants in 21 genes. The validation protocol included sequencing of cell lines (n=3) and patient samples (n=36) on an Illumina MiSeq platform. A read depth ≥100x was observed for &gt;97% of targeted bases. After filtering for artifacts, a specificity of 100% was obtained. A detection limit of ≤5% was observed for variants present in cell lines. On average two reportable variants were present in samples from patients with a myeloid neoplasm. In conclusion, the custom NGS assay provides an adequate routine assay for genetic analysis of variants present in myeloid neoplasms. Practical considerations on choice of targeted genes, type of assay and method of data analysis are provided in this report.</p

Validation and implementation of a custom 21-gene panel next-generation sequencing assay for myeloid neoplasms

Rapid and reliable mutational analysis of myeloid neoplasms is increasingly important for diagnostic, prognostic and therapeutic reasons. In this article we describe the development and validation of a custom next-generation sequencing (NGS) assay that reliably tests across a broad range of myeloid neoplasms, including AML, MDS, and myeloproliferative neoplasms. The lllumina TruSeq Custom Amplicon panel was designed to detect variants in 21 genes. The validation protocol included sequencing of cell lines (n=3) and patient samples (n=36) on an Illumina MiSeq platform. A read depth ≥100x was observed for &gt;97% of targeted bases. After filtering for artifacts, a specificity of 100% was obtained. A detection limit of ≤5% was observed for variants present in cell lines. On average two reportable variants were present in samples from patients with a myeloid neoplasm. In conclusion, the custom NGS assay provides an adequate routine assay for genetic analysis of variants present in myeloid neoplasms. Practical considerations on choice of targeted genes, type of assay and method of data analysis are provided in this report.</p

Validation and implementation of a custom 21-gene panel next-generation sequencing assay for myeloid neoplasms

Rapid and reliable mutational analysis of myeloid neoplasms is increasingly important for diagnostic, prognostic and therapeutic reasons. In this article we describe the development and validation of a custom next-generation sequencing (NGS) assay that reliably tests across a broad range of myeloid neoplasms, including AML, MDS, and myeloproliferative neoplasms. The lllumina TruSeq Custom Amplicon panel was designed to detect variants in 21 genes. The validation protocol included sequencing of cell lines (n=3) and patient samples (n=36) on an Illumina MiSeq platform. A read depth ≥100x was observed for &gt;97% of targeted bases. After filtering for artifacts, a specificity of 100% was obtained. A detection limit of ≤5% was observed for variants present in cell lines. On average two reportable variants were present in samples from patients with a myeloid neoplasm. In conclusion, the custom NGS assay provides an adequate routine assay for genetic analysis of variants present in myeloid neoplasms. Practical considerations on choice of targeted genes, type of assay and method of data analysis are provided in this report.</p

A Novel Rho-Like Protein TbRHP Is Involved in Spindle Formation and Mitosis in Trypanosomes

Background: In animals and fungi Rho subfamily small GTPases are involved in signal transduction, cytoskeletal function and cellular proliferation. These organisms typically possess multiple Rho paralogues and numerous downstream effectors, consistent with the highly complex contributions of Rho proteins to cellular physiology. By contrast, trypanosomatids have a much simpler Rho-signaling system, and the Trypanosoma brucei genome contains only a single divergent Rho-related gene, TbRHP (Tb927.10.6240). Further, only a single RhoGAP-like protein (Tb09.160.4180) is annotated, contrasting with the.70 Rho GAP proteins from Homo sapiens. We wished to establish the function(s) of TbRHP and if Tb09.160.4180 is a potential GAP for this protein. Methods/Findings: TbRHP represents an evolutionarily restricted member of the Rho GTPase clade and is likely trypanosomatid restricted. TbRHP is expressed in both mammalian and insect dwelling stages of T. brucei and presents with a diffuse cytoplasmic location and is excluded from the nucleus. RNAi ablation of TbRHP results in major cell cycle defects and accumulation of multi-nucleated cells, coinciding with a loss of detectable mitotic spindles. Using yeast two hybrid analysis we find that TbRHP interacts with both Tb11.01.3180 (TbRACK), a homolog of Rho-kinase, and the sole trypanosome RhoGAP protein Tb09.160.4180, which is related to human OCRL. Conclusions: Despite minimization of the Rho pathway, TbRHP retains an important role in spindle formation, and henc

The Ordered Extension of Pseudopodia by Amoeboid Cells in the Absence of External Cues

Eukaryotic cells extend pseudopodia for movement. In the absence of external cues, cells move in random directions, but with a strong element of persistence that keeps them moving in the same direction Persistence allows cells to disperse over larger areas and is instrumental to enter new environments where spatial cues can lead the cell. Here we explore cell movement by analyzing the direction, size and timing of ∼2000 pseudopodia that are extended by Dictyostelium cells. The results show that pseudpopod are extended perpendicular to the surface curvature at the place where they emerge. The location of new pseudopods is not random but highly ordered. Two types of pseudopodia may be formed: frequent splitting of an existing pseudopod, or the occasional extension of a de novo pseudopod at regions devoid of recent pseudopod activity. Split-pseudopodia are extended at ∼60 degrees relative to the previous pseudopod, mostly as alternating Right/Left/Right steps leading to relatively straight zigzag runs. De novo pseudopodia are extended in nearly random directions thereby interrupting the zigzag runs. Persistence of cell movement is based on the ratio of split versus de novo pseudopodia. We identify PLA2 and cGMP signaling pathways that modulate this ratio of splitting and de novo pseudopodia, and thereby regulate the dispersal of cells. The observed ordered extension of pseudopodia in the absence of external cues provides a fundamental insight into the coordinated movement of cells, and might form the basis for movement that is directed by internal or external cues

Assessment of Inactivating Stop Codon Mutations in Forty Saccharomyces cerevisiae Strains: Implications for [PSI+] Prion- Mediated Phenotypes

The yeast prion [PSI+] has been implicated in the generation of novel phenotypes by a mechanism involving a reduction in translation fidelity causing readthrough of naturally occurring stop codons. Some [PSI+] associated phenotypes may also be generated due to readthrough of inactivating stop codon mutations (ISCMs). Using next generation sequencing we have sequenced the genomes of two Saccharomyces cerevisiae strains that are commonly used for the study of the yeast [PSI+] prion. We have identified approximately 26,000 and 6,500 single nucleotide polymorphisms (SNPs) in strains 74-D694 and G600 respectively, compared to reference strain S288C. In addition to SNPs that produce non-synonymous amino acid changes we have also identified a number of SNPs that cause potential ISCMs in these strains, one of which we show is associated with a [PSI+]-dependent stress resistance phenotype in strain G600. We identified twenty-two potential ISCMs in strain 74-D694, present in genes involved in a variety of cellular processes including nitrogen metabolism, signal transduction and oxidative stress response. The presence of ISCMs in a subset of these genes provides possible explanations for previously identified [PSI+]-associated phenotypes in this strain. A comparison of ISCMs in strains G600 and 74-D694 with S. cerevisiae strains sequenced as part of the Saccharomyces Genome Resequencing Project (SGRP) shows much variation in the generation of strain-specific ISCMs and suggests this process is possible under complex genetic control. Additionally we have identified a major difference in the abilities of strains G600 and 74-D694 to grow at elevated temperatures. However, this difference appears unrelated to novel SNPs identified in strain 74-D694 present in proteins involved in the heat shock response, but may be attributed to other SNP differences in genes previously identified as playing a role in high temperature growth

Cytochrome P-450 2D6 and 2C19 polymorphisms and consumption of care in psychiatric practice

Background: Cytochrome P-450 2D6 (CYP2D6) and 2C19 (CYP2C19) are known to contain functional polymorphisms that can alter the metabolic rate of many antidepressants (AD) and antipsychotics (AP). Individuals with no metabolic activity and with increased activity may be at risk for an unsatisfactory drug response. Objectives: Aim of this study was to assess the influence of CYP2D6 and CYP2C19 phenotypes on the consumption of care in psychiatric patients. Methods: The study was conducted in a psychiatric hospital in the Netherlands, including all admissions from July 2001 until July 2010, of patients genotyped for (at least) the CYP2D6 ∗3 ∗4 and gene multiplication and CYP2C19 ∗2. Patients were classified as poor metabolizers (PM), ultrarapid metabolizers (UM), intermediate metabolizers (IM) and extensive metabolizers (EM). To study the consumption of care, several outcome measures were calculated for PM and UM and compared to EM. Results: 7377 admissions were analyzed, belonging to 3859 unique patients. The total duration of hospital stays with AP and/or AD treatment was longer for CYP2D6 UM than for CYP2D6 EM (66 versus 48 days,