A pathogenic mosaic TP53 mutation in two germ layers detected by next generation sequencing

Abstract

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.

Figure 1

A. Mutant read frequency in different samples. The patient's blood samples were all taken around the time of neuroblastoma diagnosis. Numbers of sequencing reads per sample are displayed in the top level. * p = 0.01, comparing mutant read count frequencies between samples PD9058b3 and PD9058b with Fisher's exact test. Differences between PD9058b2 and sample b/b3 are not significant (p>0.05). B. Capillary sequencing of control germline DNA (Reference sequence) and patient's blood samples extracted at three time points, showing very low level of adenine (black arrow). The guanine (black peak) dropped approximately between 8–17% relative to control, as given by the software, which would not usually be classed as significant when looking for heterozygous germline mutations. C and D. Immunohistochemistry to show strong nuclear staining p53 in neuroblastoma (C) and sarcoma NOS (D).