Identification of a novel TP53 cancer susceptibility mutation through whole-genome sequencing of a patient with therapy-related AML

Abstract

Context: The identification of patients with inherited cancer susceptibility syndromes facilitates early diagnosis, prevention, and treatment. However, in many cases of suspected cancer susceptibility, the family history is unclear and genetic testing of common cancer susceptibility genes is unrevealing.

Objective: To apply whole-genome sequencing to a patient without any significant family history of cancer but with suspected increased cancer susceptibility because of multiple primary tumors to identify rare or novel germline variants in cancer susceptibility genes. DESIGN, SETTING, AND PARTICIPANT: Skin (normal) and bone marrow (leukemia) DNA were obtained from a patient with early-onset breast and ovarian cancer (negative for BRCA1 and BRCA2 mutations) and therapy-related acute myeloid leukemia (t-AML) and analyzed with the following: whole-genome sequencing using paired-end reads, single-nucleotide polymorphism (SNP) genotyping, RNA expression profiling, and spectral karyotyping.

Main outcome measures: Structural variants, copy number alterations, single-nucleotide variants, and small insertions and deletions (indels) were detected and validated using the described platforms. RESULTS; Whole-genome sequencing revealed a novel, heterozygous 3-kilobase deletion removing exons 7-9 of TP53 in the patient's normal skin DNA, which was homozygous in the leukemia DNA as a result of uniparental disomy. In addition, a total of 28 validated somatic single-nucleotide variations or indels in coding genes, 8 somatic structural variants, and 12 somatic copy number alterations were detected in the patient's leukemia genome.

Conclusion: Whole-genome sequencing can identify novel, cryptic variants in cancer susceptibility genes in addition to providing unbiased information on the spectrum of mutations in a cancer genome.

Figure 1. TP53 germline deletion

A. Shown is the number of sequence reads per 100bp of genomic DNA that mapped to the TP53 genomic locus. Changes in sequence read depth indicate a heterozygous and homozygous deletion of TP53 in the skin and bone marrow samples, respectively (upper panel). The deletion includes exons 7-9 of TP53 (based on transcript ID ENST00000269305); genomic coordinates of the deletion boundaries are shown (lower panel). B. Genomic DNA isolated from the patient’s skin or bone marrow (BM) or maternal blood were amplified by PCR using the two primer sets depicted in Figure 1A (lower panel). The first primer set (marked “1”) produces a 2,924 bp product from the wild type but not mutant TP53 allele. The second primer set (“2”) is predicted to amplify 4,169 bp and 1,179 bp products from the wild type and mutant TP53 alleles, respectively. However, due to its smaller size, only the mutant band was consistently seen. C. TP53 RNA expression as determined by RNA expression profiling using Affymetrix Exon 1.0 arrays. The probe signal value for each exon is plotted from the t-AML patient along with the signal for 6 AML samples without TP53 mutations. Box and whisker plots for the 6 AML samples are shown; the black dots represent the t-AML sample. There are 2 probe sets for exon 7. D. RT-PCR of the patient’s bone marrow RNA was performed using primers in exons 6 and 11 of TP53. The wild type and mutant TP53 transcripts produced the predicted bands of 614 bp and 204 bp (asterisk), respectively. E. Sequencing of the mutant band demonstrated the in-frame splicing of exon 6 to exon 10 (beginning with “ATC”). Tick marks represent the placement of each nucleotide.

Figure 2. The TP53 deletion allele produces a TP53 protein lacking transcriptional activity

A. Immunostaining for TP53 of bone marrow from our patient or a healthy donor. Original magnification 40X. B. Human SaOS2 cells were transfected in duplicate with 250 ng wild-type TP53-responsive p50-2Luc promoter-reporter and either 100 ng CMV-Neo (Neo; negative control without TP53 expression), wild-type TP53 (WT), t-AML patient’s TP53 (tAML) or hot spot DNA binding mutant p53-R175H (175H; known non-binding mutant control). Expression levels of TP53 protein were determined by Western blot. “FL” refers to the full-length protein, and “Δ” is the mutant t-AML patient’s protein. Blots were probed for β-actin as a loading control. C. Transactivation of the TP53-responsive p50-2Luc promoter, represented as relative light units (RLU), was determined 48 hours after transfection. Similar results were obtained with transfection of a higher amount (1µg) of the TP53 expression constructs (data not shown). D. Expression of well-defined TP53 target genes as determined by RNA expression profiling using Affymetrix Exon 1.0 arrays. The probe signal value for the t-AML sample (black bars) and the mean signal ± SEM of 6 AML samples without TP53 mutations (grey bars) are shown.

Figure 3. Somatic mutations and structural variants in the t-AML genome

A. Mutational spectrum in the t-AML leukemic genome compared to 2 de novo AML genomes without TP53 mutations. Shown on the X axis are the various possible nucleotide transitions and transversions; the Y axis represents the percentage of mutations across the genome with that type of mutation. B. Validated somatic SNVs in coding genes in the t-AML patient. Shown is the frequency of sequence reads for the mutated allele (compared with total sequence reads) for bone marrow and skin DNA. C. Spectral karyotyping of the patient’s leukemic blasts. Chromosomal rearrangements are boxed. D. A t(3;4) reciprocal translocation resulted in the fusion of DGKG (chromosome 3; light grey) and BST1 (chromosome 4; dark grey). There is also a duplication of exons 10-14 of DGKG. Shown is a schematic of the translocation breakpoint and the primers used to confirm expression of the fusion transcripts by RT-PCR. The DGKG-BST1 fusion results in the splicing of exon 14 of DGKG with exon 6 of BST1(starting with “TAT”). The primers used to confirm expression of this fusion by RT-PCR are indicated with circles with the number 1. The BST1-DGKG fusion results in the splicing of exon 5 of BST-1 with exon 10 of DGKG (starting with “GGC”). The primers used to confirm expression of this fusion by RT-PCR are indicated with circles with the number 2. Sequencing of the PCR products showed that both fusion genes were expressed in-frame. Tick marks represent the exact placement of the nucleotides, and letters below the arrow represent the amino acid symbol.