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ABSTRACT
Non-syndromic cleft lip with or without cleft palate (NSCLP), the most common human craniofacial malformation, is a complex disorder given its genetic heterogeneity and multifactorial component revealed by genetic, epidemiological, and epigenetic findings. Epigenetic variations associated with NSCLP have been identified; however, functional investigation has been limited. Here, we combined a reanalysis of NSCLP methylome data with genetic analysis and used both in vitro and in vivo approaches to dissect the functional effects of epigenetic changes. We found a region in mir152 that is frequently hypomethylated in NSCLP cohorts (21–26%), leading to mir152 overexpression. mir152 overexpression in human neural crest cells led to downregulation of spliceosomal, ribosomal, and adherens junction genes. In vivo analysis using zebrafish embryos revealed that mir152 upregulation leads to craniofacial cartilage impairment. Also, we suggest that zebrafish embryonic hypoxia leads to mir152 upregulation combined with mir152 hypomethylation and also analogous palatal alterations. We therefore propose that mir152 hypomethylation, potentially induced by hypoxia in early development, is a novel and frequent predisposing factor to NSCLP.
Acknowledgments
We are thankful to the Passos-Bueno lab members for helpful discussions and lab organization. We thank Patrícia Semedo Kuriki for helping with cell sorting. We also express our gratitude to Vanessa Naomi and Daiane Gil Franco for the sequencing services and for performing the RNA-seq run during the COVID-19 pandemics. We also thank Dale Bryant for the support with this manuscript. This work was supported by FAPESP/CEPID 2013/08028-1, FAPESP 2017/11430-7 (LA), 2016/23648-4 (LAB), and CNPq 305405/2011-5 (MRPB) research fellowships.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability
The DOI or other location of our data 10.4121/19181903
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592294.2022.2115606
