“mir152hypomethylation, potentially triggered by embryonic hypoxia, as a common mechanism for non-syndromic cleft lip/palate”

Abstract

12 13 Non-syndromic cleft lip/palate (NSCLP), the most common human craniofacial 14 malformations, is a complex disorder given its genetic heterogeneity and multifactorial 15 component revealed by genetic, epidemiological and epigenetic findings. Association of 16 epigenetic variations with NSCLP has been made, however still of little functional investigation. 17Here we combined a reanalysis of NSCLP methylome data with genetic analysis and used both 18 in vitro and in vivo approaches to dissect the functional effects of epigenetic changes. We found 19 a frequent differentially methylated region in mir152, hypomethylated in NSCLP cohorts (21-20 26%), leading to mir152 overexpression. In vivo analysis using zebrafish embryos revealed that 21 mir152 upregulation leads to craniofacial impairment analogue to palatal defects. Also, we 22 demonstrated that zebrafish embryonic hypoxia leads to mir152 upregulation combined with 23 mir152 hypomethylation and also analogue palatal alterations. We therefore suggest mir152 24 hypomethylation, potentially induced by hypoxia in early development, as a novel and frequent 25 predisposing factor to NSCLP. 26 molecular alteration for complex traits 42 . Here, we attempted to identify both group and 53 individual-specific methylation changes using previously published methylome data on NSCLP. 54We identified individual methylation changes in known NSCLP candidate regions and also 55 mir152 hypomethylation in 26% of our discovery cohort. This result was replicated in an 56 independent cohort and validated through functional in vitro and in vivo assays. Finally, we 57 demonstrated how hypoxia, a known environmental risk factor for NSCLP, can modulate such 58 changes. 59 60Results 61 62 mir152 is a frequent differentially methylated region in the Brazilian NSCLP cohort 63 64We conducted differential methylation analysis at the gene level using the whole 65Brazilian NSCLP 450K dataset (66 NSCLP vs 59 controls 24 , and looked for the top 5 DMRs 66 ranked by RnBeads, which combines adjusted p-value to methylation difference and 67 methylation quotient. Those top DMRs were, in order of ranking: top 1, an intronic region of 68 CROCC at 1p36.13; top 2, an intronic region of FAM49B at 8q24.21; top 3, an intronic region of 69 NLK at 17q11.2; top 4, a non-coding region comprising mir152 at 17q21.32; and top 5, an 70 exonic region of PRAC2 and comprising mir3185 also at 17q21.32 (Figure 1a; Supplementary 71 Table 2). Among those genes, mir152 (adjusted p-value= 8.20E-06, beta-difference = -0.04) 72 was the only with enriched expression during palatal embryogenesis in human and mouse, 73 according to Sysface (Systems tool for craniofacial expression-based gene discovery) online 74 tool. Moreover, mir152 has already been identified as a DMR during normal murine palatal 75 development 53 and suggested as a central regulator of downstream mRNAs encoding proteins 76 known to play pivotal roles in orofacial development 54 , however still with no clear evidence of 77 association with NSCLP. Concur...