Detection and Functional Analysis of TP53 Mutations in CLL

Methods Mol Biol. 2019;1881:63-81. doi: 10.1007/978-1-4939-8876-1_6.

Abstract

Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).

Keywords: FASAY; Low-burden mutations; Next-generation sequencing; Subclonal mutations; TP53.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • DNA Mutational Analysis / instrumentation
  • DNA Mutational Analysis / methods
  • Genes, Reporter / genetics
  • High-Throughput Nucleotide Sequencing / instrumentation
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Leukemia, Lymphocytic, Chronic, B-Cell / blood
  • Leukemia, Lymphocytic, Chronic, B-Cell / genetics*
  • Leukemia, Lymphocytic, Chronic, B-Cell / pathology
  • Mutation
  • Neoplastic Cells, Circulating / pathology
  • Saccharomyces cerevisiae / genetics
  • Transfection / instrumentation
  • Transfection / methods
  • Tumor Suppressor Protein p53 / genetics*

Substances

  • TP53 protein, human
  • Tumor Suppressor Protein p53