patricia kannouche
TRANSCRIPT
Altération des ADN polymérases: Impact sur la stabilité
génétique et effets pléiotropiques
Gustave Roussy Cancer CampusLaboratory of Genetic Stability and Oncogenesis
CNRS-UMR 8200Villejuif, France
Patricia Kannouche
Carrefour de PathologieSymposium SFP/INSERM
Anomalies de la réparation et de la réplication de l’ADN: des bases fondamentales aux applications cliniques
Palais des Congrès, Jeudi 7 novembre 2019
Presentation
I- Overview on DNA replication
II- Identification and characterization of somatic and germinal mutations
in POLE or POLD1 genes in tumors
III- Non-cancer diseases with germinal mutations in DNA polymerase genes
Epidemiological data showed that DNA replication errors may account for two-
thirds of the mutations found in human cancers (Tomasetti et al., Science 2015,
2017)
Classical DNA polymerases and associated subunits
➢ The human genome encodes at least 14 DNA polymerases — a surprisingly large number !
DNA polymerases in human genome
Adapted from Loeb and Monnat, 2008
Pola Pold Pole Polg
Déterminants de la fidélité de la réplication
Adapté de McCulloch and Kunkel, Cell Res 2008
G
T
parentalnéosynthétisé
GMSH2/MSH6
MLH1/PMS2
G
T
EXO1
G
PCNA polδ
reconnaissance
excision
resynthèse
Fidélité de la réplication de l’ADN
Taille du génome humain: env 3 . 109 paires de base
Adapté de McCulloch and Kunkel, Cell Res 2008
G
T
parentalnéosynthétisé
GMSH2/MSH6
MLH1/PMS2
G
T
EXO1
G
PCNA polδ
reconnaissance
excision
resynthèse
Déterminants de la fidélité de la réplication
12
➢ Tumors with somatic mutations in exonuclease domain of DNA polymerase epsilon exhibit overall mutation frequencies often exceeding 100 mutations/Mb.
➢ Tumors with somatic mutations in the exonuclease domain of DNA polymerase delta, or deficient in MMR : 10-100 mutations/Mb.
➢ Tumors with somatic mutations in the exonuclease domain of DNA polymerase epsilon and deficient in MMR (bMMRD) : ~ 600 new mutations/generation.
DNA replication-associated errors are repaired by two components: polymerase proofreading and mismatch repair.
Déterminants de la fidélité de la réplication
Campbell et al, Cell 2017
Replication repair deficiency drives a mutator phenotype
in many adults cancers
New hereditary colorectal cancer syndrome
“Polymerase Proofreading Associated Polyposis” (PPAP)
➢ Germline pathogenic variants in human POLE or POLD1 exonuclease domains
➢ Multiple polyposis and CRC following an autosomal dominant pattern of
inheritance
➢ Contributions of POLD1 and POLE genetic defects to early onset
of familial colorectal cancer: 0.2 and 0.6%, respectively
➢ Larger phenotypic spectrum :
- endometrial cancer
- ovarian and brain tumors
- pancreatic and small intestine cancer
- melanoma.
Rayner et et al, Nature Review Cancer 2016
POLEPOLD1
POLE and POLD1 mutations reported in CRC and EC
➢ POLE is altered much more frequently than POLD1 in hypermutated MSS tumors
➢ Most somatic POLD1 mutations are found in MSI tumors.
➢ Some mutations are observed at a vastly greater frequency than others.
POLE
POLD1
POLE P286R/P301R has a robust DNA polymerase activity
superior to that of the wild-type or proofreading-deficient yPolε
Xing et al, Nature Comms 2019
Biochemical studies on POLE P286R
POLE P286R/P301R retains weak 3ʹ→5ʹ exonuclease activity
Model explaining the ultramutator phenotype in tumors harboring the
cancer-associated P286R mutation in POLE
Xing et al, Nature Comms 2019
Relationship between the incidence of individual POLE variants in sporadic
tumors and their mutator effects deduced from in vivo functional assays
P236R
F367S
P236H
S459F
D275VL424V
P436R
Barbari, et al., 2018
Adapted from Poulos et al., PLoS Genet. 2018
POLE p.P286R
Accumulation of mutations related to Signature 10
100-fold increase in C>A transversions in T[C>A]T context
30-fold increase in C>T transitions in T[C>T]G context
Serine to Tyrosine/ Leucine
Arginine to Isoleucine/Glutamine
Glutamic Acid to Stop Codon
Strong bias for particular amino acid changes
0%
10%
20%
30%
Distinctive pattern of missense/truncation mutations in
oncoproteins and tumour suppressors
PTEN pR130Q
MSH6 pE946*
ARID1A pR1989*PIK3CA pR88Q
APC pQ1338* P53 pR213*
Mutational signatures in POLE tumors
It is extremely important to distinguish among causative, non-polymorphic germline variants, and
somatic pathogenic, non-passenger variants with prognosis meaning.
Landscape of drivers and passengers in POLE and POLD1 genes
IMAGe syndrome
Clinical features closely resembling IMAGe syndrome (associated with GOF mutations
in CDKN1C)
Intrauterine growth restriction [IUGR]
Metaphyseal dysplasia
Adrenal hypoplasia congenita
Genitourinary anomalies in males
Distinctive facial features
Variable immune dysfunction
Intronic variant (c.1686+32C>G, altering splicing) as part of a common haplotype, in
combination with different loss-of-function variants in trans.
Biallelic mutations in POLE
Logan et al, AJHG 2018
This mutation causes a distinct multisystem disorder that includes:
-subcutaneous lipodystrophy,
-deafness,
-mandibular hypoplasia
-hypogonadism in males
-Progeroid features
-Insulin resistance
MDPL syndromeAn in-frame deletion at the polymerase active site of POLD1 causes a
multisystem disorder with lipodystrophy
Clinical characteristics of individuals with MDPL syndrome
no detectable polymerase activity, but
robust exonuclease activity
Biochemical characteristics
Fibrosis
Weedon et al., Nature Genetics 2013
To date, about 25 cases with POLD1-related MDPL syndrome have been reported worldwide, albeit almost entirely of European originMale-to-female ratio 7:15. Most of patients with p.Ser605del, 3 patients with p.Arg507Cys, 1 patient with novel mutation p.Ile1070Ans, 2 related female patients with Glu1067Lys
Heterozygous mutation in POLD1
Polg, l’ADN polymérase mitochondriale
Small circular DNA
A schematic diagram of a mitochondrial DNA
replication intermediate
Copeland’s Lab