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TP53
A Comprehensive Study of TP53 Mutations in Chronic Lymphocytic Leukemia: Analysis of 1,287 Diagnostic CLL Samples Sona Pekova, MD., PhD. Chambon Ltd., Laboratory for molecular diagnostics, Prague, Czech Republic 25.6.2013, Konference DNA analýza X, Novotel Praha TP53 „The Guardian of the Genome“ (David Lane, 1992) • mapped to 17p • G1/S arrest vs apoptosis • physiologically low level of TP53 • activity of TP53 tightly regulated (MDM-2, ATM, ATR, p19ARF…) Functional analysis of TP53 Genotype does not necessarily need to translate into phenotype FASAY –Functional Analysis of Separated Alleles in Yeast • in vitro test of the functional activity of TP53 DNA-binding domain • color of transgenic yeast colonies indicates mutated or wild type TP53 allele The principle of FASAY • in vitro recombination of TP53 into GM yeasts • Selection medium with low adenine p53 promotor ADE2 • Red colonies mut pSS16 • White colonies wt p53 p53 respons promotor Red colonies are re-sequenced to identify clonal TP53 mutation A. Detection of biologically relevant TP53 variants C277Y 60.1 % red colonies B. Identification of small subclones with TP53 mutation E220R 27.5 % red colonies C. Splicing TP53 variants „delta ex6“ TP53 splicing variant in CLL 45.3 % red colonies on FASAY Pekova et al., Leukemia Research, 2010 Bourdon et al., Genes Dev., 2005 Protein expression of the D ex6 TP53 variant Pekova et al., Leukemia Research, 2010 In vitro growth properties of stableDex6 TP53 cell lines H1299 Dex6 Pekova et al., Leukemia Research, 2010 H1299 mock Growth curves of stable Dex6 TP53 and mock transfected H1299 cell lines Pekova et al., Leukemia Research, 2010 Expression profiles of Dex6 TP53 and mock transfected H1299 cell lines Dex6 vs mock Affymetrix GeneChip Human Exon 1.0 ST array Pekova et al., Leukemia Research, 2010 In vitro properties of the Dex6 TP53 variant: cyclins (A1, G1, G2, F, I, B2, A2, T2) matrix metalloproteinases serine proteases hyaluronidases inhibitors of caspases adhesion molecules molecules of intercellular matrix Accented proliferative phenotype, loss of intercellular contacts, defects of apoptosis Pekova et al., Leukemia Research, 2010 D. Identification of temperature-sensitive TP53 variants S127F Mr. Brother and Mrs. Sister Case FASAY: wild type p53 FASAY: 51.8% t.s. colonies Pekova et al., Leukemia Research, 2010 Back-tracking the t.s. variant R283C, cDNA sequencing Pekova et al., Leukemia Research, 2010 Pekova et al., Leukemia Research, 2010 Site-directed mutagenesis/ Megaprimer Eco RV Eco RI C/T T T Pekova et al., Leukemia Research, 2010 Production of H1299 stable cell lines harboring temperature-sensitive TP53 variants Pekova et al., Leukemia Research, 2010 Stable H1299 cell lines expressing temperature-sensitive variants of TP53 • Temperature-sensitive TP53 variants V157F, A161T, S215I, V216M, Y234C resemble in vitro R175H • Temperature-sensitive TP53 variants N235S, R283C, K320E, K320T resemble in vitro WT TP53 Pekova et al., Leukemia Research, 2010 List of all TP53 mutations/variants identified in the study Mutation Nr. Mutation Nr. Mutation Nr. Mutation Nr. E68X W91X K101E P109I R110L F113S Y126C Y126H K132N Q136E A138V K139R C141Y Q144X W146X P151S P152L P153L R156P V157F V157G A159P A161T 3 1 1 1 2 2 2 1 2 1 1 2 1 2 1 1 1 1 1 2 1 2 3 K164E Q167X V172A V173A R175H C176R C176W P177L H178R P178S H179L H179R R181S D186N G187D L188P P190H P190S H193L H193R L194P R196X Y205C 1 1 1 2 2 2 1 1 1 1 1 4 1 1 1 1 1 1 2 2 1 1 1 H214R S215I V216M R219I Y220C Y220H P222L S227P H233R Y234C Y234D Y234H N235S M237I N239D S241C M243T G245D G245S N247D R248Q R248W R249S 1 1 1 1 4 1 1 1 4 5 3 1 2 2 1 1 1 1 8 1 1 8 3 P250L P250S I251V I255T T256P E258G E258K D259G D259S G266R R267P R267W R273C R273H R273S C275G C275Y A276V C277F C277Y R280K R280P R280T 1 1 1 2 1 1 1 2 1 1 1 1 2 4 1 1 1 1 1 2 1 1 1 Y163H 2 R213X 1 R249W 1 D281G 1 Table 1: List of all TP53 mutations/variants identified in the study. Temperature-sensitive variants are highlighted in blue. Mutation D281V R282W R283C T284S E286G K305M K320E K320T L330P c.302_del c.328_339del c.503_ 578del c.504_578del c.515_559del c. 550_576del c. 636del c.704_709del c.716_736del c.724_739del c.749_751del c.792_794del c.828del c. 532_ 549del TP53 splicing variant Beta Nr. 2 1 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 30 Mutation Nr. TP53 splicing variant Delta Ex6 c.780_781insAGT c.559_560insG c.782_783insTAT c.102_103insC 16 1 1 1 1 Summary • 1,287 diagnostic CLL samples tested • 18,4% of FASAY-corroborated TP53 mutations/variants • Modes of TP53 inactivation diverse (point mutations, insertions, deletions, temperature-sensitive variants, aberrant splicing variants) • Splicing variants might play biological role • Temperature-sensitive TP53 variants V157F, A161T, S215I, V216M, Y234C resemble in vitro R175H • Temperature-sensitive TP53 variants N235S, R283C, K320E, K320T resemble in vitro WT TP53 Pekova et al., Leukemia Research, 2010 Thank you for your attention www.chambon.cz
