With an estimated incidence of nearly 300.000 new cases per year and a mortality rate around 50%, oral cancers are major challenge in oncology and represent the sixth leading cancer worldwide, according to GLOBOCAN report 2018 1.

The main risk factors are exposure to genotoxic agents such as tobacco, alcohol and betel nut chewing 234.These agents induced genetic alteration in genes controlling the cell cycle such as TP53567. Also, poor oral hygiene, bad set of teeth, inadequate diet and poor nutrition, or immunosuppression, may increase oral cancer risk by promoting chronic infections of the oral mucosa with high risk Human Papilloma Virus (HPV), by inducing loss of function of TP53 encoded protein 8910.

Mutations of TP53 gene have been reported as the main driver event of carcinogenesis 11. Missense variants have been reported in the coding region of the DNA Binding Domain of p53 protein which specifically binds to the promoters of targeted genes.

Eight hotspot amino-acid substitutions in the DNA Binding Domain of p53, characterizing almost 27% of mutant proteins, were identified in human cancers while they are not directly cancer associated (Arg175His, Gly245Ser, Arg248Gln, Arg248Trp, Arg249Ser, Arg273His, Arg273Ser, and Arg282Trp) 12. In mice model, the introduction of the Arg172His mutation corresponding to human Arg175His, has been associated with the functional inactivation of p63 and p73 proteins 1312.

Other variants are located in a 1kb region spanning intron 3 to intron 4 containing the 6 cancer-related variants reported in IARC TP53 database 14: a 16 bp insertion in intron 3 (NG_017013.2:g.16185_16200Ins, rs59758982), two variants located in exon 4 coding for the Proline-rich domain of p53 (Pro47Ser : NG_017013.2:g.16321C>T, rs1800371) and Pro72Arg : NG_017013.2:g.16397C>G, (rs1042522)), and 2 variants located in intron 4 (NG_017013.2:g.17032T>C (rs1794287) and NG_017013.2:g.17198G>A (rs35850753) (Figure 1).

Effect predictions of these cancer related variants in Varsome database have shown benign effect (rs1800371, rs78378222 and rs35850753) or uncertain significance (rs59758982, rs1042522 and rs1794287), although functional/association studies are in favour of cancer predisposition (Table 1).

The 16 bp insertion in intron 3 (rs59758982) has been associated with increased risk of colorectal cancer in a case-control study and correlated with a reduced level of TP53 mRNA in lymphoblastoid cell-lines 15. The Pro47Ser variant (rs1800371) has been reported in populations of African ancestry (allelic frequency estimated at 2-4% in Africa and 1.2% in African-Americans) 16 and have been associated with increased risk of breast cancer among pre-menopausal women 17. The Pro72Arg (rs1042522) is a non-conserved amino acid with reported altered electrophoretic mobility, conformation and function of themutant protein 18. This variant shows significant ethnic bias with Arginine allelic frequencies estimated at ~72% for Europeans compared to ~38% for African and African-Americans and ~51% for Asian populations (gnomAD database: https://gnomad.broadinstitute.org/variant/17-7579472-G-C?dataset=gnomad_r2_1). In a previous study, we showed in an ethnicity-stratified meta-analysis that this variant is associated with oral cancers risk in Asian population (OR = 1.31; CI_95% =1.09 - 1.58; p=0.004) whereas any significant association was observed in Africans and Caucasians 19.

Two other cancer-related variants (rs1794287) and (rs35850753) are located in intron 4 and were shown to affect the activity of TP53 intron 4 internal promoter by changing its affinity with several transcription factors 20. The last variant (rs78378222) maps to the polyadenylation signal located in 3´ UTR of TP53. This variant is in linkage disequilibrium with rs35850753 in intron 4 and both have been associated with n euroblastoma (rs35850753: OR = 2.7, CI_95% = (2.0 - 3.6); rs78378222: OR = 2.3, CI_95% = (1.8 - 2.9) 21.

In Sub-Saharan Africa, very few studies have investigated genetic variability of TP53 and its association with cancer risk. Here we screened for TP53 genomic variants predisposing to oral cancers in patients living in Senegal.

The study was carried out from January 2018 to September 2021 through a collaboration between the Department of Stomatology and Maxillofacial Surgery of Aristide Le Dantec Hospital in Dakar for patients recruitment, the Senegalese National Blood Transfusion Center for the recruitment of healthy blood donor controls, the department of Human Genetics of the Faculty of Medicine, Pharmacy and Odontology of Cheikh Anta Diop University Dakar for DNA extraction and PCR amplification, and the Functional Genomics Platform of the National Center for Scientific and Technical Research (CNRST) of Rabat (Morocco) for Sanger sequencing of PCR products. The research protocol was approved by the Ethics Committee of Cheikh Anta Diop University under reference 0320/2018/CER/UCAD.

Clinical characteristics of Senegalese patients diagnosed with oral cancers have been described by Touré et al in 2005 and highlighted early age at diagnosis (52.6 years), a sex ratio in favor of females (0.67), a negative alcohol-tobacco status (62.9% non-smokers and 83.8% non-alcoholics) and a poor oral hygiene 24. This profile is quite similar to what observed in our patients. These results highlighted that tobacco and alcohol are not the common risk factors and emphasized the hypothesis that exposure to HPV and genetic factors may be associated with oral carcinogenesis in Senegalese population.

The predominant tumor histology in our patients was squamous cell carcinoma. This type represents 95% of all oral cancers tumors worldwide and is characterized by altered proliferation of dysplastic squamous cells on the surface of the epithelial layer ) 12. In mice model, the introduction of the Arg172His mutation corresponding to human Arg175His, has been associated with the functional inactivation of p63 and p73 proteins 1312.

Among oral cancer etiological factors genetic alterations in specific genes controlling the cell cycle such as TP53 have been reported. In this study, 52.27% of patients carried at least one mutation in TP53 (46/88), similar to data from Poeta et al in the US (53.3%, 224/420) 29 and Yamamoto et al in Japanese patients (49.5%, 91/194) 30.

We identified eleven TP53 variants in our patients including ten Single Nucleotide Variants (SNV) and one Copy Number Variation (CNV). Among these variants, four have been newly identified in this study. They were not reported in any variant database (ClinVar, dbSNP, Varsome…). Effect prediction by SIFT and PolyPhen tools showed possible clinical significance of 2 missense variants on p53 function (c.62A>C (p.Asp21Ala); c.773A>T (p.Glu258Val)). Further studies are required to assess their functional significance.

The other seven variants identified in this study have been reported in Clinvar, dbSNP and gnomAD databases. Two of them are the most frequent (p.Pro72Arg, rs1042522) and the 16 bp insertion (rs59758982). They are both cancer related-variant and their functional impact in p53 have been reported 14. The first one (p.Pro72Arg, rs1042522) has been extensively studied as a potential risk factor for the development of malignancies and is the most common variant associated with predisposition to oral cancers. Allelic and genotypic distribution of rs1042522 are known depending on geographic latitude and ethnicity throughout the world 3132333435. The variant has been associated in case/control studies with a higher risk of oral, nasopharyngeal, lung, thyroid, skin, cervical, prostate, bladder, gastric, colorectal, and hepatic cancers 2036373839. Arg72 allele frequency was estimated at 31.26% in our patients, similar to frequencies observed in African and African American populations as reported in gnomAD database (31.4% and 32.1% respectively). In contrast, frequencies reported for Asian and Caucasian populations are higher (58.4% and 73.6% respectively) confirming the ethnic and geographic bias observed in the distribution of this variant throughout the world.

For the 16 bp insertion in intron 3 (rs59758982), its implication on carcinogenesis has also been highlighted in several cancers: colorectal, breast, cervical, gastric, head and neck, lung, bladder, esophagus, and in glaucoma 204015. Allelic frequency of 12% has been reported in Caucasian diagnosed with colorectal cancer 40 whereas in our study, the frequency was 2 times higher (26.25%). In contrast this variant was not detected in 425 Japanese patients with primary open angle glaucoma 41.

Association study between these two cancer-related variants did not find any significant association in our population as we previously reported in a meta-analysis 19. However, LD test showed that they are strongly linked and could be transmitted together in a common haplotype predisposing towards oral cancers. It is therefore necessary to study the haplotype structure and investigate the role of neighboring variants spanning intron 3 to intron 4 region, especially intron 4 where an internal promoter encoding nine p53 isoforms has been located. Mutations in this promoter have been reported to affect the transcription and expression level of p53 isoforms 2042. Eiholzer and colleagues have recently demonstrated in 2020 that this genomic region harbor several haplotypes blocks that could increase the level of expression of the Δ133_TP53 transcript, which at certain levels of expression can have pro-tumor effects 43. Therefore it would be of interest to investigate by long read target sequencing and functional analysis this genomic region in patients diagnosed with cancer, and definitely address its implication on carcinogenesis.

Our study highlighted that despite the absence of association between the two most common cancer-related variants of TP53 observed in Senegalese patients diagnosed with oral cancer, their strong LD suggests that they are transmitted together in a common haplotype and may be implicated in oral carcinogenesis. Further functional and genomic studies are needed to explore the implication of the genomic region spanning intron 3 to intron 4 of TP53 gene where most cancer-related variants are located.