TP53 c.799C > T maps to a highly conserved region of exon 8 that encodes the DNA binding domain of p53 (amino acids 102-292) (Figs. 3f,g), by which p53 targets genes that control cell cycle arrest (such as CDKN1A, GADD45, PLK3, and MDM2) and apoptosis (such as BAX). This missense variant is predicted to be damaging by SIFT but only possibly damaging by Polyphen2, and ClinVar has conflicting interpretations of pathogenicity, from uncertain significance to likely pathogenic.13 Classification of this variant as likely pathogenic is supported by well-established in vitro functional studies. In yeast, human p53 p.Arg267Trp retained at least partial transactivation activity of p53-responsive elements including BAX, GADD45, and MDM2 but had a loss of p21 binding.6,14 In human glioblastoma cells, p53 p.Arg267Trp reduced activation of BAX and CDKN1A reporters by 22 and 23%, respectively, compared to wild type p53 (ref. 15). Similarly, p53 p.Arg267Trp did not induce transcription of CDKN1A and PLK3 reporters, among others, or cell death in response to external stimuli in human TP53 null colorectal carcinoma cells.16 Taken together, these data suggest that c.799C > T results in partial loss-of-function.

TP53 c.799C > T is not listed in the Exome Aggregation Consortium browser or the Genome Aggregation Database but has been reported in the literature as a germline variant in three independent studies. These include an early-onset female breast cancer patient17 and a woman age 52 years with multiple tumors (breast fibroadenoma, subependymoma, melanoma in situ, and sessile serrated adenoma)18 who were heterozygous for c.799C > T. A third individual age 43 years was a compound heterozygote for c.799C > T and c.665C > T (p.Pro222Leu) and had no personal history of cancer.19 The Saudi family in this report had no history of breast cancer or melanoma, but similar to the third study,19 had three individuals (III.2, IV.3, and IV.5) heterozygous for c.799C > T with no personal history of cancer. Of the three affected individuals in this study, the proband and his oldest sister (IV.1) had CPC diagnosed at a very young age while the proband’s paternal aunt (III.1) had liver cancer at age 49 years. This spectrum of phenotypes and variable penetrance could be due to the remaining transactivation activity of the TP53 protein product. Alternatively, recent work has identified genetic events that modify the LFS phenotype including intragenic variants, variants of genes in the p53 regulatory pathway, telomere attrition, and copy number variation.9 In the targeted 30-gene panel used for genetic testing, we did not identify any large structural variants such as copy number variation in any of the family members who received NGS testing. Our future studies will include analyses of whole genome sequencing of the proband’s nuclear family to identify other candidate variants.

Heterozygous germline pathogenic variants are reported in approximately 70% of classic LFS families,20 and homozygosity for germline TP53 pathogenic variants is extremely rare. To our knowledge, there have only been five previous reports of homozygous germline TP53 pathogenic variants. Four of the reports were of children in south and southeast Brazil who had the TP53 c.1010G > A (p.Arg337His) Brazilian founder allele; three children had ACC,21,22,23 and one child had CPC.23 The fifth report is of homozygous truncating pathogenic variants in a child age 2 years with CPC and rhabdomyosarcoma.24 In stark contrast, in this study, the proband’s father is homozygous for c.799C > T and presents phenotypically normal at 39 years with no personal history of cancer. By age 50 years, men with LFS have a 68% risk of developing cancer, with an average age of onset at 40 years.25,26 However, those estimates are based on studies of men with heterozygous variants. Given the spontaneous tumors and premature death observed in P53 knockout mouse models,27 we would expect that the presence of two pathogenic TP53 alleles in humans would lead to a compound phenotype with increased risk for early onset cancer. It is possible that the proband’s father is germline heterozygous with a somatic variant in his peripheral blood lymphocytes, which could be attributed to classic mosaicism or clonal hematopoiesis associated with aging.28,29 However, we did not observe any evidence of retention of the reference allele by NGS (AF at 100%, 297×) or Sanger sequencing (Figs. 3d, e). Unfortunately, we have been unable to obtain additional tissue samples to further test this hypothesis.

Based on the analysis of this single family, we hypothesize that TP53 c.799C > T has low or variable penetrance for LFS, with predisposition to the development of CPC. CPC accounts for 1 to 4% of all pediatric brain tumors but is frequently found in families with LFS.9 Recent reports estimate that between 36 to 63% of individuals with CPC carry a TP53 pathogenic variant.9,30,31,32 Based on family history and imaging records, we suspect that the proband’s great-grandmother’s (presumed carrier) and paternal grand-aunt’s brain tumors were also CPCs. CPC is extremely rare in adults, however, there have been several recently reported diagnoses in individuals ranging from ages 21 to 73 years.33,34,35 We will continue to follow-up with the proband’s two unaffected carrier sisters and his paternal aunt (III.3) for development of CPC.

CPC has a high incidence of recurrence and metastasis along the central nervous system, and children with TP53-immunopositive CPC, as is the case with the proband and his oldest sister, have a five-year survival rate of 0% compared to those with TP53-immunonegative CPC at 82% (ref. 9). With no established curative therapy, intensive surveillance protocols can significantly improve long-term survival rates in children and adults. These protocols use a combination of physical exams, blood tests, and imaging for early tumor detection and reduction of cancer, and a recent panel of LFS experts recommended that surveillance should be offered to all individuals carrying a pathogenic TP53 variant and individuals who fit the classic definition of LFS, regardless of TP53 status.36 As our understanding of TP53 function and phenotype-genotype correlation in LFS continues to expand, targeted molecular therapies hold the greatest promise for preventing tumor recurrence and prolonging survival. For example, a recent case report demonstrated successful treatment of a 4-month-old female who presented with a recurrent and metastatic CPC through molecular-guided therapy. After 36 months of treatment, her MRI showed 92% tumor reduction, and the metastatic tumor was cleared; she continued to thrive one year after completion of the study.37 Targeted therapy options such as this will continue to expand through genetic modeling of LFS and utilization of recently developed methodologies.38

In summary, this is the first detailed report of a family with the extremely rare TP53 missense variant c.799C > T. The younger age of onset and increased disease severity with successive generations suggests the possibility of genetic anticipation, which has been reported in LFS families with TP53 pathogenic variants.39,40,41 Further analysis are needed to understand how this may influence the mechanism of disease in this family. However, the observations from this family have furthered our understanding of the phenotypic variability that may be caused by one variant of TP53, even in the same family, and suggest that other factors (genetic and/or environmental) may play a role in mechanism of disease manifestation in LFS.