Congenital microcephaly is caused by a multitude of drivers affecting maternal−fetal health during pregnancy. It is a rare outcome in high-income industrial countries where microcephaly rates are in the range of 0.3−0.9 per 1000 newborns. Prevalence of microcephaly varies considerably across developing countries and can go as high as 58 cases per 1000 live births in pregnancies exposed to infection by Zika virus (ZIKV). Not only ZIKV-infected pregnancies, but other drivers can modulate the occurrence and severity of this outcome. Here, we sought to test the ZIKV−microcephaly association vs. competing hypotheses using a meta-analysis with 8341 microcephaly cases pooled from 10,250,994 newborns in the Americas, Africa, and Asia. Analysis of risk ratios (RR) showed teratogens the most likely microcephaly-associated risk factor (RR = 3.43; 95%-CI 2.69−4.38; p-value < 0.0001), while the statistical significance of the ZIKV−microcephaly association was marginal (RR = 2.12; 95%-CI 1.01−4.48; p-value = 0.048). Other congenital infections showed strong but variable associations with microcephaly (RR = 15.24; 95%-CI 1.74−133.70; p-value = 0.014). Microcephaly cases were associated with impoverished socioeconomic settings, but this association was statistically non-significant (RR = 2.75; 95%-CI 0.55−13.78; p-value = 0.22). The marginal ZIKV−microcephaly association and statistical significance of the competing hypotheses suggest maternal ZIKV infection might not be a cause of microcephaly alone.

Zika virus (ZIKV)-associated congenital microcephaly is an important contributor to pediatric death, and more robust pediatric mortality risk metrics are needed to help guide life plans and clinical decision making for these patients. Although common etiologies of pediatric and adult mortality differ, early life health can impact adult outcomes-potentially through DNA methylation. Hence, in this pilot study, we take an early step in identifying pediatric mortality risk metrics by examining associations of ZIKV infection and associated congenital microcephaly with existing adult DNA methylation-based mortality biomarkers: GrimAge and Zhang\'s mortality score (ZMS).
Mortality measures were calculated from previously published HumanMethylationEPIC BeadChip data from 44 Brazilian children aged 5-40 months (18 with ZIKV-associated microcephaly; 7 normocephalic, exposed to ZIKV in utero; and 19 unexposed controls). We used linear models adjusted for chronological age, sex, methylation batch and white blood cell proportions to evaluate ZIKV and mortality marker relationships.
We observed significant decreases in GrimAge-component plasminogen activator inhibitor-1 [PAI-1; β = -2453.06 pg/ml, 95% confidence interval (CI) -3652.96, -1253.16, p = 0.0002], and ZMS-site cg14975410 methylation (β = -0.06, 95% CI -0.09, -0.03, p = 0.0003) among children with microcephaly compared to controls. PAI-1 (β = -2448.70 pg/ml, 95% CI -4384.45, -512.95, p = 0.01) and cg14975410 (β = 0.01, 95% CI -0.04, 0.06, p = 0.64) results in comparisons of normocephalic, ZIKV-exposed children to controls were not statistically significant.
Our results suggest that elements of previously-identified adult epigenetic markers of mortality risk are associated with ZIKV-associated microcephaly, a known contributor to pediatric mortality risk. These findings may provide insights for efforts aimed at developing pediatric mortality markers.