Zika virus (ZIKV) is a human teratogen that infects neural cells of children exposed during pregnancy and causes a spectrum of multiple congenital anomalies named Congenital Zika Syndrome (CZS), which includes microcephaly, brain calcifications, lissencephaly, ventriculomegaly, ocular alterations, among others (del Campo et al., 2017). It has been reported that up to 42% of all individuals exposed to ZIKV during pregnancy indeed develop the CZS (Nithiyanantham and Badawi, 2019).

Recently, in silico and in vitro studies have shown that the p53 protein, encoded by the TP53 gene, is differentially expressed in human neuroprogenitor cells (hNPCs) during ZIKV infection (El Ghouzzi et al., 2016; Zhang et al., 2016). Importantly, the increase in the TP53 expression under this condition induce cell cycle arrest and death of developing neurons, and it has been proposed as a possible mechanism for ZIKV teratogenesis (El Ghouzzi et al., 2016; Bhagat et al., 2018). Through a systems biology approach, it was identified that p53 is the central protein of a genetic regulatory network of proteins associated with the ZIKV infection and those associated with microcephaly occurrence (Teng et al., 2017). In addition, it was demonstrated that increased p53 activity leads to an increased death rate of neural cells due to the binding of the ZIKV capsid protein to MDM2 protein, decreasing its activity as one of the main p53 negative regulators (Teng et al., 2017).

In a context of wild-type TP53 sequence, constitutive p53 protein levels and activity can be modulated directly or indirectly by several regulator proteins and specific microRNAs (miRNAs) (Liu and Chen, 2006; Hermeking, 2012). LIF, a cytokine encoded by the LIF gene, is a p53 regulator that can both indirectly repress p53 functions in human cells through the induction of MDM2, or it can be up-regulated by p53 itself (Liu et al., 2015). Like p53, LIF protein appears to be increased in brain cells infected by ZIKV (Bayless et al., 2016). Recently, the role of miRNAs has been described in ZIKV infection, and dysregulation of nervous system development pathways mediated by several miRNAs was demonstrated (Bhagat et al., 2018). The miR-605 is an miRNA that acts as an indirect modulator of TP53 gene expression through the negative regulation that it exerts targeting MDM2 transcripts (Xiao et al., 2011).

Based on previous evidence of differential susceptibility to ZIKV teratogenesis in humans, as well as possible involvement of the p53 signaling pathway in this outcome, we investigated the role of functional single nucleotide variants (SNVs) in genes of the p53 signaling pathway as potential susceptibility factors to CZS in a sample of Brazilian children exposed to ZIKV during pregnancy. In addition, we evaluated the impact of ZIKV infection on the expression of genes of this pathway in neuroprogenitor cells, the main targets of ZIKV in the developing brain.

The clinical and sociodemographic description of the sample used in this study was previously performed in another study by our group (Gomes et al., 2021). However, we summarize here in Table 1 the clinical, gestational, and sociodemographic characteristics of both study groups. Mothers of children with CZS had ZIKV infection predominantly in the first trimester of pregnancy (80%) while mothers of children without congenital anomalies reported infection predominantly in the third trimester (44%) (p<0.001). Mothers of children with CZS exhibited a lower educational level (p<0.001) and lower monthly family incomes compared to mothers of children without congenital malformations (p=0.008). Children without CZS had higher weight, height and cephalic perimeter measures compared to with CZS.

Congenital anomalies observed in children with CZS also were previously described in the aforementioned study (Gomes et al., 2021), but they are summarized in Figure 1 . Detailed dysmorphologic, ocular and imaging exams were not carried out on all individuals with CZS, since some research and assistance centers had limited resources to perform such tests. The most prevalent clinical findings were microcephaly (n=40, 100%) brain calcifications (n​​=2/24, 92%), cerebral atrophy (n=11/12, 92%), lissencephaly (n=10/12, 83%), craniofacial disproportion (n=15/18, 83%), ventriculomegaly (n=8/11, 73%), and ocular alterations (n=22/32, 69%).

Figure 2 provides a summary of the proposed relationships among genes and SNVs studied here and their interplay in regulating the p53 pathway. Regarding the allelic and genotypic frequencies of the SNVs selected in TP53, MDM2, MIR605 and LIF, they were not statistically different between CZS and control groups ( Supplementary Table 2 ). Comparing the allelic frequencies obtained in the case and control groups with the frequencies reported in the Brazilian and worldwide population, we observed that they were quite similar ( Supplementary Table 2 ), but it is important to note that the Brazilian population is highly admixed (Kehdy et al., 2015), and the allelic and genotypic frequencies of the our population are not always similar to other specific populations.

Allelic frequencies were also compared, within the group of children with CZS, between those who presented or not specific brain malformations, such as intracranial calcification, lissencephaly, cerebral atrophy, hydrocephaly and ocular alterations (information available for more than 12 individuals) ( Supplementary Table 3 ). A significative difference in the allelic frequencies of the SNV TP53 rs1042522 was found between individuals with CZS and lissencephaly or without lissencephaly. Interestingly, we detected the G allele with a frequency lower than expected in individuals with lissencephaly and much higher than expected in individuals without lissencephaly (p=0.007) ( Table 2 ). Genotypic frequencies were also different between groups, but this difference was tangential to the significance.

Gene expression analysis showed an increased expression of LIF in human neuroprogenitor cells three days after the infection by the Asian strain of ZIKV, but this association lost its significance after correcting the p-value (logFC = 1.41, p = 0.001, FDR = 0.44). The other genes did not have their expression disturbed by the ZIKV infection ( Supplementary Table 4 ).

The ability of ZIKV to impair the central nervous system development is still poorly understood. Although it is known that only up to 42% of individuals exposed to ZIKV develop CZS, genetic components related to this susceptibility have not yet been recognized (Nithiyanantham and Badawi, 2019). In this study we assessed the role of selected SNVs in p53 pathway genes as potential susceptibility factors to CZS in a sample of Brazilian children exposed to ZIKV during pregnancy. Besides, we evaluated how ZIKV infection deregulates the expression of the same genes in neuroprogenitor cells.

The p53 signaling pathway was chosen for investigation in this study because it has been shown to be affected by ZIKV during infection (El Ghouzzi et al., 2016; Tang et al., 2016; Teng et al., 2017). Similarly, the dysregulation of miRNAs in human neuronal cells during ZIKV infection has also been reported by several studies (Bhagat et al., 2018). Considering these findings and the well-documented miRNA network acting in the direct and indirect regulation of the p53 pathway, we also evaluated in this work an SNV in a miRNA that has been shown to positively regulate p53 activity (Xiao et al., 2011; Hermeking, 2012).

Although we did not find an association between the studied SNVs and susceptibility to CZS, we found a lower frequency of individuals with CZS and lissencephaly carrying the G-allele of the TP53 rs1042522 while children without lissencephaly had this allele in homozygosis. Lissencephaly comprises a group of disorders characterized by an abnormally smooth surface of the cerebral cortex due an abnormal neuronal migration, where genes related to the neural migration process are usually mutated (Mochida, 2009). The G-allele of the TP53 rs1042522 has been associated with higher p53 activity and more potent capacity to induce apoptosis (Dumont et al., 2003). During ZIKV infection, p53 expression has already been shown to be upregulated in an attempt to decrease viral proliferation and eliminate the virus (El Ghouzzi et al., 2016; Tang et al., 2016). In this context, individuals carrying the G allele could have an advantage in viral elimination, with more efficient apoptosis, while those carrying the C allele would allow the virus to spread on a larger scale, impairing the differentiation and activity of a greater number of neural cells. Despite being biologically consistent with the development of an altered phenotype, such as lissencephaly, this result should be interpreted with caution, since this association was identified in a small subset of patients. Therefore, additional case-control studies with a larger sample size and extensive clinical characterization are required to confirm this finding.

The ability of ZIKV infection to affect the expression of p53 pathway genes (El Ghouzzi et al., 2016; Zhang et al., 2016) was one of the main reasons that led to the development of this study. Thus, to evaluate how ZIKV affected the expression of TP53, MDM2, MIR605 and LIF in hNPCs, we performed an analysis of differential gene expression in exposed cells compared to unexposed cells. The study used for such analysis (Liu et al., 2019 – GSE129180) was different from those cited as a reference previously (El Ghouzzi et al., 2016 – GSE78711; Zhang et al., 2016 – GSE80434). In this analyze, we found only the upregulation of LIF due to ZIKV infection, not corroborating the previously reported findings on TP53 and MDM2 (El Ghouzzi et al., 2016; Zhang et al., 2016) and not identifying an alteration in the MIR605 expression. However, it is important to mention that although the cell type evaluated by these three studies was the same (hNPCs), the differences in the lineage of the ZIKV(Asian or African) and the time of exposure to ZIKV have a strong influence in which genes are affected and how significant is the altered expression. In this sense, we saw that in the study by El Ghouzzi and colleagues (2016) the analysis of the differential gene expression occurred 72 hours after infection by the Asian strain of ZIKV, while in the study by Zhang et al. and colleagues (2016) the evaluation was 64 hours after infection by the Asian strain, and in the study by Liu et al. (2019) the analysis occurred 3 days (72h) and 6 days after infection by the Asian lineage, which means that no study has used the same methodological approaches and, consequently, different results can be expected.

Regarding the gestational and socio-environmental features, the first trimester of ZIKV infection has been associated with the development of more severe congenital anomalies and abortions, while the socioeconomic level, closely related to nutritional and health conditions, could be discussed as a possible risk factor for ZIKV teratogenesis (França et al., 2016; Barbeito-Andrés et al., 2020). In this sense, our study corroborated the previous findings on increased risk to CZS due to ZIKV exposure during the first trimester (França et al., 2016). In addition, our findings suggest that socioeconomic level may be an important risk factor, which deserves to be further explored in the context of ZIKV teratogenesis.

Lastly, this study has some limitations that must be considered in the interpretation of the results, such as the sample size, which affects our power to identify strong associations or to perform more robust statistical analyses. The size of our complete sample is small (n = 88) and when subgroup comparisons are made, it becomes even smaller. Thus, considering our sample size and based on studies that have already performed comparisons of allelic frequencies of polymorphisms in samples of individuals exposed to ZIKV during pregnancy (Santos et al., 2019; Gomes et al., 2021), the power of the present study to identify significant associations was approximately 40%. In this sense, we highlight the importance of future studies being carried out on larger samples, making it possible to confirm the associations found by this study, as well as carrying out more robust statistical analyzes, such as the allele size effect. Currently, it is especially difficult to increase the sample size, since fortunately the ZIKV outbreak in Brazil has decreased since 2017. However, it is important to emphasize that this is one of the most largest cohort of children exposed to ZIKV during pregnancy used in the context of studies on genetic susceptibility to ZIKV teratogenesis (Caires-Júnior et al., 2018; Candelo et al., 2019; Santos et al., 2019; Aguiar et al., 2020; Gomes et al., 2021). The lack of detailed data on gestational history and clinical description for some patients is also a limitation of this study, motivated by the regional variations in available resources during medical consultation. The different origin of the CZS and control groups may also constitute a confounding factor in the study, because the number of cases from each Brazilian region was not matched with the same number of controls for the same region, and, considering that the Brazilian population is a very admixed, it is reasonable to speculate that differential genetic variability between groups might have been observed for that reason and may not strictly related to the development of CZS.

In conclusion, although some studies have described that p53 pathway genes seems to be affected by ZIKV infection and they need attention given to their essential functions in embryonic and fetal development and apoptosis, in the present study we did not find any association between selected variants in genes of this pathway and an increased risk to ZIKV teratogenesis. Importantly, we identified a possible association between TP53 rs1042522 and the occurrence of lissencephaly in CZS patients, which should be further explored in additional studies. We emphasize that, although biological pathways involved in the teratogenesis of ZIKV have been reported in recent years, additional efforts are still needed to elucidate the main genetic factors implicated in the susceptibility to CZS. Additional case-control and functional studies focusing on both SNVs located in p53 pathway genes and in specific miRNA genes regulating ZIKV-mediated teratogenesis should be performed on individuals exposed to ZIKV in order to better understand their role in the CZS development.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

This study involved human participants and, therefore, was reviewed and approved by Ethics and Research Committee of the Hospital de Clínicas de Porto Alegre - n° 170619 – CAAE 78735817910015327. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

JAG, LS-F, and FSLV: conceptualization of the study. JAG, ES, IAV, ACPTT, JHS, BFRR, MFG, TMO, MDFCA, and IFC: acquisition, analysis, and interpretation of the data. JAG: writing—original draft preparation. JAG, LRF, PA-P, LS-F, and FSLV: writing—review and editing. JAG, LS-F, and FSLV: supervision, project administration and funding acquisition. All authors contributed to the article and approved the submitted version.

This work was supported by the Instituto Nacional de Genética Médica Populacional (INAGEMP) [grant number CNPq 573993/2008-4, FAPERGS 17/2551.0000521-0], Fundo de Incentivo a Pesquisa e Eventos (FIPE) of the Hospital de Clínicas de Porto Alegre (HCPA) [grant numbers 2017-0619, 2019-0295], Conselho Nacional de Desenvolvimento Científico e Tecnológico [grant numbers 424362/2018-0 and Chamada CNPq/CAPES/MS-14/2016]. FSLV is recipient of a CNPq scholarship grant [grant number CNPq 312993/2017-0]. PA-P is recipient of a CNPq scholarship grant [grant number CNPq 307826/2017-1].

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.