We present here the clinical phenotype, demographic and genetic characteristics of carriers of the TP53, p.Arg181Cys variant of Arab-Muslim descent treated at our center with a focus on 30 cancer patients and their corresponding pedigrees. The combination of the cancer types, variable age at onset, and discrepancy in family history of malignancy, with 15 of 24 families meeting updated Chompret criteria for LFS, supports a decreased penetrance and an attenuated phenotype for this TP53 variant. Nevertheless, the majority of cancer patients (25 of 30) in this cohort were diagnosed before the age of 50 including four carriers who were diagnosed with primary CNS tumors in childhood. Furthermore, no additional familial, demographic, environmental or genetic factors were found that may explain the differences in age of onset of cancer. The lack of other factors which can assist in the prediction of the degree of penetrance amongst carriers is in-line with previous analyses conducted on carriers of attenuated TP53 variants [13]. We therefore recommend that carriers of TP53 p.Arg181Cys follow strict surveillance and early detection tests akin to those recommended in classical LFS, specifically the use of annual whole body and brain MRI from infancy. Future analysis of genetic modifiers, somatic alterations, and clinical data and by age group, may allow for a more accurate understanding of age-associated risks, which could ultimately inform age-adjusted surveillance protocols.

Considering that the TP53 p.Arg181Cys variant was found exclusively in the Arab-Muslim population, the majority of which originated from of the Jerusalem or Hebron area, the shared haplotype, together with the fact that this variant has rarely been described outside this geographical area, we suggest that TP53 p.Arg181Cys is a founder mutation predominant to the Arab-Muslim population in this area. While attenuated LFS is well described in the context of ever-increasing genetic testing in cancer patients, the finding of a pathogenic variant causing an attenuated phenotype together with a founder effect in large number of carriers (30 cancer patients and 21 healthy carriers) isolated to a specific region and population, has been rarely described outside the Brazilian and Jewish-Ashkenazi variant [35, 36]. In light of its increased frequency among the sub-population of Arab patients in Israel, TP53 p.Arg181Cys was included in 2020 as one of the pathogenic variants tested as part of the founder mutations panel recommended by the Israeli Ministry of Health to all Arab-Muslim breast cancer patients, regardless of their age at diagnosis. We suggest broadening this recommendation to all patients of Arab-Muslim descent diagnosed with cancer characteristic of LFS of, as well as reaching out to family members of known carriers of TP53 p.Arg181Cys for cascade genetic testing. Healthy carriers identified can then be recommended the appropriate surveillance regimen. Such cascade testing and subsequent screening has already benefited at least two patients in this cohort diagnosed with early-stage breast cancer as a result of incidental identification of the variant in non-cancer patients referred for genetic testing due to developmental delay.

Notably, TP53 p.Arg181Cys has been described in one other pediatric case—a 1 year old boy with rhabdomyosarcoma, who was later diagnosed with adrenocortical carcinoma and osteosarcoma at 2 years of age, as a result of whole body MRI surveillance, demonstrating the importance of genetic screening for TP53 p.Arg181Cys and subsequent surveillance in high-risk population [6]. This cohort is the first to report the TP53 p.Arg181Cys variant in several very young pediatric cases, and numerous cases in the early adulthood, further warranting genetic screening of this this high-risk population and strict surveillance protocols for all established TP53 p.Arg181Cys carriers.

The TP53 p.Arg181Cys variant resembles the TP53 p.Arg337His founder variant detected in one of every 300 individuals in Southeastern Brazil. Similar to our cohort, while initially reported in cases of pediatric adrenocortical carcinoma patients, large-scale studies have since described breast cancer as the most common tumor in carriers of TP53 p.Arg337His with later onset in comparison to classical LFS [35, 37]. Additionally, p.Arg337His confers a highly variable cancer risk, ranging from individuals who remain unaffected over their lifetime to those who meet the Chompret Criteria for LFS. This variability was not found to be influenced by demographic factors or carcinogenic exposures but rather mediated by the co-inheritance other pro-apoptotic tumor suppressors, such as XAF1 p.E134* [35]. Finally, current surveillance and screening recommendation for TP53 p.Arg337His are in line with those suggested for classical LFS and have found to be both efficient in reducing cancer mortality and cost-effective [38].

The TP53 p.Arg181Cys variant also resembles the TP53 p.Gly334Arg variant reported in 22 cancer patients from 16 families predominantly of Jewish-Ashkenazi descent in Northern America. Though 4 of 22 cancer patients were diagnosed with pediatric adrenocortical carcinoma, the most common tumor among carriers of TP53 p.Gly334Arg was breast cancer (10 of 22 patients) with relatively late onset (range 30–65 years old). Additionally, while most probands reported a family history of cancer, only 6 of 16 probands in which ancestry was available met updated Chompret Criteria for LFS thus, providing evidence of reduced penetrance in a TP53 variant distinct to a specific population [36].

The fact that two young cancer patient in our cohort are homozygous for TP53 p.Arg181Cys is intriguing and supports the partial oncogenic properties of this variant. While over 90% of carriers of TP53 who develop LFS-associated tumors, exhibit somatic loss of the wild-type allele [39], germline bi-allelic variations, including homozygosity, have so far been described in only a handful of cases. Most of these were in carriers of the Brazilian variant, and all presented with early onset of cancer, albeit limited penetrance among their family members [40,41,42]. While a recent haplotype analysis of 38 unrelated carriers of TP53 p.Arg337His found two homozygous patients with early onset of cancer (ages 6 and 9 years old) with a compound TP53 p.Arg337His and XAF1 p.E134* haplotype, other reports did not rule out additional co-variants[43]. WES done on one homozygous patient in our cohort did not identify additional variations in known TP53 modifiers. Hence, it remains unclear if homozygosity, or rather the presence of other co-variants leads to increased penetrance among these homozygous patients.

The TP53 181 codon is a H1 helix residue is located in the apoptosis-stimulating of P53 protein 2-domain (ASPP2). While structural models have shown this codon to be essential for dimer stability, modification of TP53 181 retains partial protein function. TP53 p.Arg181 modifications in cancer-cell lines and mouse models have shown to be able to activate p21CDKN1A or MDM2 at levels similar to wild-type TP53, but unable to activate genes associated with apoptosis, such as NOXA or p53AIP1 [44, 45]. TP53 p.Arg181Cys in mice demonstrated a modest increase in cancer incidence and increased lipolytic activity implicated in cancer development [46]. Several functional analyses of an array of TP53 variants expressed in yeast or mammalian cells, which were recently shown to be highly correlated, determine TP53 p.Arg181Cys to be an outlier variant with partial loss of apoptotic activity. These assays also demonstrate that while TP53 p.Arg181Cys loses some tumor suppressor functions, it does not cause additional oncogenic gain of function (GOF) which is typical to TP53 variant ‘hotspots’ associated with classical LFS [47,48,49]. Recently, Landau et al. implemented a more continuous computational approach to determine TP53 pathogenicity, using tumor variant amplitude, in which TP53 p.Arg181Cys was shown be a weak oncogenic driver [50]. Finally, analyses of other variants causing attenuated LFS, mainly the Brazilian variant, have shown cancer susceptibility to be dependent on co-inheritance of other proapoptotic tumor suppressors involved in TP53 mechanism [35]. These animal models and functional and computational analyses may explain the pathogenicity and reduced penetrance of TP53 p.Arg181Cys, demonstrated in our clinical findings.

Cancer patients in our cohort had relatively good clinical outcomes. While TP53 mutations have shown mixed impact on clinical outcomes in cases of breast cancer, TP53 mutations are surrogate for markedly improved prognosis in CNS tumors [51, 52]. As previously mentioned, TP53 p.Arg181Cys has limited penetrance and a phenotype resembling attenuated-LFS, by retention of several wild-type properties which relegate it a weak oncogene driver when compared to pathogenic variants in hotspots causing classical LFS. Previous studies, both clinical and computational, have shown a correlation between the degree of penetrance of attenuated-LFS and survival [4, 7, 50].

Interestingly, both cancer patients and healthy carriers had limited co-morbidities, were in good general health and had low BMI. These findings hint to potentially favorable properties of TP53 p.Arg181Cys, which may contribute to the good clinical outcomes. A previous in-vivo analysis of muscle specimens taken from volunteers after exercise, demonstrate that healthy carriers of TP53 variants, have better muscle recovery, improved recovery of phosphocreatine and enhanced mitochondrial function compared to healthy non-carriers. This was substantially more prominent for the 7 carriers of TP53 p.Arg181Cys included in the study, in which levels of mitochondrial respiratory complex proteins associated with biogenesis were significantly increased [25]. TP53 p.Arg181Cys knock-in mice show a uniquely high proportion of lean muscle tissue, increased aerobic exercise endurance, increased lipolytic activity and transactivation of genes involved in fatty-acid metabolism which do not appear in knock-in models of other TP53 variants [46]. In addition, haplotype analysis conducted in this cohort show that TP53 p.Arg181Cys appeared in conjunction with the p.Arg72 SNP in both cancer patients and healthy carriers. While this SNP has limited impact on cancer risk, data from cell lines and mice models show that this SNP markedly affects the response of P53 to nutrient alterations, driving increased inflammation in mice on a high-fat diet and alters the ability of mutant P53 to bind and inhibit the PGC-1α metabolism regulator thus inducing cancer-promoting metabolism [53]. Our clinical results support this in-vivo evidence that TP53 p.Arg181Cys acts as a double edge sword – causing attenuated-LFS while at the same time enhancing lipolytic activity and metabolism which in turn may promote tumorigenesis. Moreover, these findings hint at the potential therapeutic approach to delay cancer development among TP53 p.Arg181Cys carriers by decreasing mitochondrial function through common anti-diabetic medication, a theory based on mouse models [54]. Finally, analysis of other attenuated LFS variants in TP53 have shown that cancer development is dependent on the development of compound mutant haplotypes including other pro-apoptotic tumor suppressors and loss of somatic wildtype TP53 alleles [39, 43].

Given the rarity of TP53 variants, our clinical data on 30 cancer patients carriers and 21 healthy carriers and their families due to a founder effect in a specific population and region, is highly valuable. Nevertheless, our study was limited since we could not complete genetic testing in all family members, nor build more detailed age-specific pedigrees is some families, which could have provided more informative data for age-associated penetrance. In addition, some of the healthy carriers identified are still young and therefore may develop cancer in the future, which will impact the ratio of affected to unaffected carriers. Long-term data should be collected from these families as the carriers age and the families expand, to further determine our findings over time and perhaps lead to a less rigid surveillance protocol.