A Pathogenic Mosaic TP53 Mutation in Two Germ Layers Detected by Next Generation Sequencing

et al. (2014) A Pathogenic Mosaic TP53 Mutation in Two Germ Layers Detected by Next Generation Sequencing. PLoS ONE 9(5): e96531. doi:10.1371/journal.pone.0096531 A Pathogenic Mosaic TP53 Mutation in Two Germ Layers Detected by Next Generation Sequencing Sam Behjati 0 Mariana Maschietto 0 Richard D. Williams 0 Lucy Side 0 Mike Hubank 0 Rebecca West 0 Katie Pearson 0 Neil Sebire 0 Patrick Tarpey 0 Andrew Futreal 0 Tony Brooks 0 Michael R. Stratton 0 John Anderson 0 Sylvie Mazoyer, Cancer Research Centre of Lyon, France 0 1 Cancer Genome Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus , Hinxton, Cambridgeshire , United Kingdom , 2 Department of Paediatrics, University of Cambridge, Cambridge, United Kingdom, 3 Unit of Molecular Haematology and Cancer Biology, UCL Institute of Child Health , London , United Kingdom , 4 Departments of Clinical Genetics, Great Ormond Street Hospital , London , United Kingdom , 5 Departments of Histopathology, Great Ormond Street Hospital , London , United Kingdom Background: Li-Fraumeni syndrome is caused by germline TP53 mutations and is clinically characterized by a predisposition to a range of cancers, most commonly sarcoma, brain tumours and leukemia. Pathogenic mosaic TP53 mutations have only rarely been described. Methods and Findings: We describe a 2 years old child presenting with three separate cancers over a 6 month period; two soft tissue mesenchymal tumors and an aggressive metastatic neuroblastoma. As conventional testing of blood DNA by Sanger sequencing for mutations in TP53, ALK, and SDH was negative, whole exome sequencing of the blood DNA of the patient and both parents was performed to screen more widely for cancer predisposing mutations. In the patient's but not the parents' DNA we found a c.743 G.A, p.Arg248Gln (CCDS11118.1) TP53 mutation in 3-20% of sequencing reads, a level that would not generally be detectable by Sanger sequencing. Homozygosity for this mutation was detected in all tumor samples analyzed, and germline mosaicism was demonstrated by analysis of the child's newborn blood spot DNA. The occurrence of separate tumors derived from different germ layers suggests that this de novo mutation occurred early in embryogenesis, prior to gastrulation. Conclusion: The case demonstrates pathogenic mosaicim, detected by next generation deep sequencing, that arose in the early stages of embryogenesis. - Funding: SB is funded through the Wellcome Trust PhD Programme for Clinicians. JA holds a Great Ormond Street Hospital Charity Leadership Award and receives support from the Great Ormond Street Hospital Biomedical Research Centre. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Germline mutations in the TP53 gene cause Li-Fraumeni syndrome (LFS, OMIM 151623), an autosomal dominant highly penetrant cancer predisposition syndrome characterized by a variety of early onset tumors [1,2,3]. LFS is associated with an increase in overall cancer incidence in affected individuals and an early age of cancer onset [4]. Many patients are affected in early childhood, most often by sarcomas, brain and adrenocortical tumors [5]. However, there are many families with a hereditary predisposition to cancer suggestive of LFS who do not meet the classical clinical diagnostic criteria and yet still carry TP53 mutations; less rigorous criteria were therefore developed [6,7,8]. Different mutant p53 proteins may have diverse functional and biological effects, which could partially explain the heterogeneity reported between Li-Fraumeni families [9]. Tumor spectrum, severity and phenotype can be associated with other germline genetic factors [10,11] and/or increase in DNA copy number variation [12,13]. While 70% of the patients diagnosed with classical LFS have mutations in TP53 [3], less than 20% of cases classified using less stringent criteria carry mutation in the gene. This raises the possibility of other causative germline gene mutations for LFS, or of patients with mosaicism for germline mutations, undetectable using current methods for TP53 screening. Identification of TP53 mutation has important clinical implications for early detection and treatment of associated neoplasms through clinical surveillance [14]. Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. The current study provides new insights into the role of mosaic genetic variants in cancer, and the use of sequencing technologies for their identification. We describe a child presenting with metastatic neuroblastoma and two soft tissue tumors within the first 2 years of life, in the absence of a family history of cancer. Germline mutation of TP53 was undetectable by conventional Sanger sequencing but identified by whole exome sequencing. This mutation was screened in three tumors, which displayed high rates of the mutated allele. Further analysis in the blood from the childs neonatal blood spot test (Guthrie card) showed the mutation was present at birth with the same mosaic pattern. Material and Methods Patient samples For using of parents and child samples, written informed consent for germline genetic analysis, including whole exome sequencing, was obtained from the parents of the index case patient in this study using standard UK clinical National Health Service consent procedures. Research Governance approval for detailed genetic analysis was provided by the Great Ormond Street Hospital Research and Development Department (approval 11MH09). DNA and RNA extraction DNA from frozen samples and Guthrie card was extracted with QIAamp DNA Mini Kit (Qiagen) following the manufacturers protocol. Paraffin blocks were cut as 8 mm sections on plain glass slides. Targeted regions for sampling were marked on adjacent hematoxylin and eosin sections by the study pathologist and recovered by scrape macrodissection. Between 3 and 20 sections were macrodissected depending on the tissue samples size. DNA from formalin-fixed paraffin embedded sections was extracted with QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturers instructions. RNA from frozen tumour sample was extracted using the RNeasy minikit (Qiagen) and cDNA synthesized using the SuperScript III (Invitrogen), using standard protocols. Whole exome sequencing Whole exome sequencing from genomic (native) DNA of the child (sample PD9058b) and his parents was performed by BGI, Shenzhen, China, on an Illumina HiSeq DNA Analyzer using Agilent SureSelect Human All Exon 50 Mb for target enrichment. Forty to fifty percent of the target region had a minimum coverage of 30X. Sequencing reads were aligned to the human genome (NCBI build 37) using the BWA algorithm on default settings. Reads which (...truncated)