Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer

Abstract

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Fig. 1. Clustering of cancer predisposing mutations in PPM1D

a, PPM1D mutations and cancer phenotype. b, PPM1D gene with region targeted by mutations (mutation cluster region) in blue; c, PPM1D protein showing position of mutation cluster region downstream of the phosphatase domain and upstream/overlapping the nuclear localisation signal (NLS); d, mutation cluster region showing position of mutations. The numbers above give the position of the mutations and correspond to the IDs in panel (a). Ov ca, ovarian cancer; br ca, breast cancer; bil br ca, bilateral breast cancer.

Fig. 2. PPM1D mutations are mosaic in lymphocyte DNA

a, Sanger sequencing traces showing mutant allele is lower in genomic DNA extracted from peripheral blood lymphocytes (gDNA) than typical for heterozygous mutations. The cDNA analysis demonstrates the mutations lead to truncated products not nmRNA decay. b, deep PCR amplicon sequencing showing heterozygous BRCA1/2 variants at 50% (open dots) whereas the PPM1D mutation is present at a lower percentage (red dots). c, Haplotype analysis in two families. The offspring of PPM1D mutation carriers have different maternal haplotypes spanning PPM1D (highlighted), but neither carry the mutation, indicating that it is either not present, or mosaic in the germline of the proband.

Fig. 3. The effect of mutant PPM1D isoforms on p53 activation

p53 wildtype U2-0S human osteosarcoma cells were transfected with PPM1D cDNA expression constructs and exposed to ionising irradiation (5 grays). At 30 minute and 4h intervals whole cell lysates were western blotted to estimate the IR-induced activation of p53. Western blots showing p53 and actin (loading control) protein levels at different times (in hours) after IR exposure are shown. ‘Empty’: transfected with empty construct, ‘PPM1D WT’: transfected with wildtype PPM1D construct, ‘PPM1D c.1384C>T’ and ‘PPM1D c.1420delC’: transfected with mutant PPM1D constructs. Suppression of p53 was enhanced in cells transfected with mutant constructs suggesting these alleles encode hyperactive PPM1D isoforms.