A Novel Protocol to Identify Mutations in Patients with Wiskott–Aldrich Syndrome
| Autor: | Eileen Remold-O'Donnell, Fred S. Rosen, Dianne M. Kenney, Jessica Cooley, L.N. Jones, Maxim I. Lutskiy |
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| Rok vydání: | 2002 |
| Předmět: |
Male
Time Factors Wiskott–Aldrich syndrome Platelet disorder DNA Mutational Analysis macromolecular substances medicine.disease_cause Polymerase Chain Reaction Exon medicine Humans Missense mutation Molecular Biology Gene DNA Primers Probability Genetics Mutation biology Wiskott–Aldrich syndrome protein Infant Newborn Infant Proteins Reproducibility of Results Exons Cell Biology Hematology medicine.disease Wiskott-Aldrich Syndrome Child Preschool Mutation testing biology.protein Molecular Medicine Wiskott-Aldrich Syndrome Protein |
| Zdroj: | Blood Cells, Molecules, and Diseases. 28:392-398 |
| ISSN: | 1079-9796 |
| DOI: | 10.1006/bcmd.2002.0523 |
| Popis: | Mutations of WASP (Wiskott-Aldrich syndrome protein) underlie the severe immunodeficiency/platelet disorder Wiskott-Aldrich syndrome (WAS) and its milder variant X-linked thrombocytopenia (XLT). The affected gene, a 12-exon structure on the X-chromosome, is expressed exclusively in blood cells. The encoded product WASP is a 502-amino-acid scaffolding protein that functions in stimulus-induced nucleation of actin filaments to form dynamic cell surface projections. To date, more than 150 mutations have been identified in 300 WAS/XLT kindred worldwide, generally through methodologies that include sophisticated exon screening steps such as single-strand conformation analysis. We report here a simpler protocol, which was designed for use in clinical settings to identify the mutations of newly diagnosed patients. The approach relies on directly sequencing amplified exons according to a staggered schedule based on statistical evaluation of previous cases. In a 2 1/2-year trial, samples from 28 consecutive patients were analyzed; these included 3 "blindly labeled" previously studied cases. The mutations that were identified include a broad spectrum (8 missense, 3 nonsense, 5 splice site mutations, 11 small insertion/deletions, 1 large deletion) and were broadly distributed (in 10 of the 12 exons). All mutations were verified and no discrepancies were encountered. Per patient, a mean of six DNA sequencing reactions and 6-7 h of staff effort sufficed for mutation identification and verification, indicating that the protocol is cost-effective. This cumulative experience demonstrates the suitability, reliability, and versatility of the new protocol. |
| Databáze: | OpenAIRE |
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