A 26-year-old woman with a history of a childhood adrenal tumor and osteosarcoma was referred for genetic testing. A heterozygous pathogenic variant in the TP53 gene was identified (from blood), which is consistent with a diagnosis of Li Fraumeni syndrome (Figure 1). Subsequently, the patient elected to undergo IVF (two cycles) with preimplantation genetic testing, which did not identify the variant in any of 10 embryos. This prompted follow-up testing in additional tissues, including a new blood sample. Next-generation sequencing identified the variant in all her tested tissues at varying allele fractions (Figure 2).
The correct answer is ...
The mother is exhibiting mosaicism for the TP53 variant.
All 10 embryos inherited the wild-type TP53 allele.
Li Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome associated with pathogenic variants in the TP53 gene. The hallmark feature of LFS is development of multiple primary cancers with an early age of onset.1,2
The probability that all 10 embryos (from two cycles of IVF) inherited the wild-type allele for TP53 is less than 0.01% (1/2^10) and therefore highly unlikely for an autosomal dominant condition where approximately 50% of offspring would be expected to inherit the pathogenic allele.
The preimplantation genetic testing assay has poor sensitivity to detect the variant in embryonic cells.
The preimplantation genetic testing (PGT) assay determines the genotype of embryos for single-gene disorders before transfer to the uterus. It typically utilizes a few cells from the trophectoderm of the embryo at the blastocyst stage, which are cells that develop into the placenta.3 While misdiagnosis is possible due to false-positive or false-negative results, this may be observed in the context of aneuploidy (number of chromosomes is too many or too few) rather than in the context of a sequence alteration, as seen in this individual. Confirmation of PGT findings is always recommended by follow-up amniocentesis or chorionic villus sampling to address this possibility.
The TP53 variant is a result of clonal hematopoiesis of indeterminate potential (CHIP), and therefore is not heritable.
CHIP is defined as an age-related phenomenon occurring in healthy individuals (without overt malignancy) and is characterized by clonal expansion of hematopoietic stem cells or progenitor cells harboring hematologic malignancy-related genetic variants in specific genes, including TP53.4 They are more likely to be present at low allele fractions, as they are confined to subpopulations of hematologic cells. As these are somatically acquired variants, they will not be inherited by progeny and they will be absent from all other tissues, the latter being the differentiating feature between CHIP and inherited disorders such as LFS. Additionally, CHIP has been associated with numerous adverse conditions including increased risk of cardiac events, development of hematological malignancies, and poor overall survival in this context. Therefore, it is important to correctly elucidate the origin of this TP53 variant because clinical and reproductive-risk implications would be quite different in the context of CHIP versus LFS.
What is mosaicism?
During embryonic development, gastrulation results in the formation of three germ layers: endoderm, mesoderm, and ectoderm, each of which differentiates into various tissues of the body. When a new genetic alteration occurs in a zygote very early on, all daughter cells will carry that variant and it will be present in all germ layers/tissues tested. As a heterozygous variant, it will be detected at a VAF of approximately 50%. Additionally, it will be absent from the individual’s parents. This is termed a de novo variant. However, when a genetic alteration occurs at a later timepoint, it will affect a smaller percentage of cells and may not be present in all germ layers/ tissues. It may also be detected at a VAF <50%, depending on the time at which the variant was acquired, and the proportion of cells affected. This is termed mosaicism.5
In the laboratory, detection of a variant below a VAF of 30% raises the suspicion of mosaicism and is typically followed up by testing of a different tissue (helps to rule out CHIP).6 Considering most genetic testing is performed on leukocytes from blood (derived from mesoderm), the subsequent tissue tested is usually skin fibroblasts (mesoderm) and/or buccal cells (ectoderm). In the current case, the TP53 variant was detected at VAF of 31.1% in blood, which is right at the threshold and therefore may have been reported as a heterozygous variant or as a mosaic variant, depending on laboratory-specific criteria.
Testing of additional tissues from the patient showed the presence of this variant in all three germ layers at different VAFs: 44.6% in saliva (ectoderm), 18.3% in fibroblasts (mesoderm), and only 9.0% in healthy colon tissue (endoderm). This finding rules out CHIP and is suggestive that the variant arose at an early enough time point during development to be present in all three germ layers, albeit not significantly affecting endoderm-derived colon tissue. Ideally, obtaining tissues from each germ layer provides a more complete understanding of the distribution of the variant; however, this type of testing is limited by the tissue that may be easily sampled. When a male partner is suspected of mosaicism, testing may be carried out on their sperm cells. Testing of female ovarian tissue, however, is invasive and not as straightforward. In the current case, the absence of the TP53 variant in 10 embryos implies that our patient’s gonadal tissue (mesoderm) is likely unaffected or harbors the variant in a small proportion of cells. It is therefore not associated with a significant reproductive risk in this individual and is the most likely explanation for 10 unaffected embryos.
Why is germline mosaicism clinically important to identify?
Identification of mosaicism is important for relaying disease and reproductive risks to patients, highlighting how these may differ from patients with classic LFS or CHIP. A de novo TP53 variant that is present in all cells of an individual will have a 50% risk of inheritance. A variant resulting from CHIP is somatic, with essentially a zero-risk of inheritance. For a mosaic variant however, counselling for risk of inheritance is challenging. A variant may be present in somatic tissues alone with complete absence from gonadal tissue, which implies a low to zero risk of inheritance, similar to this case. Conversely, a variant may arise after differentiation of the primordial germ cells (embryonic precursors of sperm and eggs). In this scenario, the variant would be absent from all other tissues and only present in gonadal tissue. Standard molecular testing in blood would thus be misleadingly negative. However, the individual would have a risk of passing on the variant to their offspring, which is dependent on how many germ cells were affected.
In conclusion, interpretation of a variant identified at an allele fraction <30% warrants several considerations, including mosaicism and CHIP.6 Analysis from another tissue type can distinguish these two scenarios. However, counselling for true risk for inheritance is challenging. In light of this uncertainty, current practice guidelines recommend the same clinical management for mosaic TP53 individuals and their families as for non-mosaic heterozygous individuals.2
Nisha Kanwar, Ph.D.
Fellow, Laboratory Genetics and Genomics
Wei Shen, Ph.D.
Consultant, Laboratory Genetics and Genomics
Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science