DNA methylation analysis of Homeobox genes implicates HOXB7 hypomethylation as risk factor for neural tube defects

Abstract

Neural tube defects (NTDs) are common birth defects of complex etiology. Though family- and population-based studies have confirmed a genetic component, the responsible genes for NTDs are still largely unknown. Based on the hypothesis that folic acid prevents NTDs by stimulating methylation reactions, epigenetic factors, such as DNA methylation, are predicted to be involved in NTDs. Homeobox (HOX) genes play a role in spinal cord development and are tightly regulated in a spatiotemporal and collinear manner, partly by epigenetic modifications. We have quantified DNA methylation for the different HOX genes by subtracting values from a genome-wide methylation analysis using leukocyte DNA from 10 myelomeningocele (MMC) patients and 6 healthy controls. From the 1575 CpGs profiled for the 4 HOX clusters, 26 CpGs were differentially methylated (P-value < 0.05; β-difference > 0.05) between MMC patients and controls. Seventy-seven percent of these CpGs were located in the HOXA and HOXB clusters, with the most profound difference for 3 CpGs within the HOXB7 gene body. A validation case-control study including 83 MMC patients and 30 unrelated healthy controls confirmed a significant association between MMC and HOXB7 hypomethylation (-14.4%; 95% CI: 11.9-16.9%; P-value < 0.0001) independent of the MTHFR 667C>T genotype. Significant HOXB7 hypomethylation was also present in 12 unaffected siblings, each related to a MMC patient, suggestive of an epigenetic change induced by the mother. The inclusion of a neural tube formation model using zebrafish showed that Hoxb7a overexpression but not depletion resulted in deformed body axes with dysmorphic neural tube formation. Our results implicate HOXB7 hypomethylation as risk factor for NTDs and highlight the importance for future genome-wide DNA methylation analyses without preselecting candidate pathways.

Keywords: DNA methylation; HOX genes; HOXB7; Myelomeningocele; epigenetics; neural tube defects.

Figure 1.

HOXB7 methylation studies by Sequenom EpiTYPER in MMC patients. (A) Localization of the studied amplicon (Chr17:46,685,144–46,685,550) within HOXB7 Exon 2. The amplicon covers 26 single CpGs and our assay provides data on 10 analytical CpG units. Nucleotide positions accord to the NCBI build 37/hg19. The CpG units studied by 450K Array (cg11041817, cg22622477 and cg07547765) and the in silico analysis (cg06493080, cg09357097) are also indicated. (B): Boxplot representing the methylation pattern of MMC patients and controls with box = 25th and 75th percentiles; bars = min and max values. The mean methylation level of each group is shown below the plot. (C): Methylation pattern for each CpG unit within the amplicon. Wilcoxon Rank-Sum test was performed. (D): Boxplot representing the methylation pattern of MMC patients and controls divided according to MTHFR 677C>T genotype with box = 25th and 75th percentiles; bars = min and max values. The mean methylation level of each group is shown below the plot.

Figure 2.

HOXB7 methylation studies by Sequenom EpiTYPER in pairs of unaffected siblings vs. MMC patients. (A) Boxplot representing the methylation pattern of affected siblings and unaffected siblings with box = 25th and 75th percentiles; bars = min and max values. The mean methylation level of each group is shown below the plot. (B) Methylation pattern for each CpG unit within the amplicon. Wilcoxon Rank-Sum test was performed.

Figure 3.

Phenotype analysis of Hoxb7a-overexpression in zebrafish embryos. (A) Phenotype analysis at 72 hpf of Hoxb7a mRNA injected zebrafish resulted in significant hypopigmentation and malformation in 66% of the injected zebrafish. These embryos had shorter anterior/posterior axes as well as crooked or bent tails. (B) Phenotype analysis after pax2a staining at 24 hpf resulted in about 63% embryos with a mild or severe affected phenotype after Hoxb7a overexpression compared to 13% in injected controls. (C) Pax2a staining after microinjection of different concentrations of mRNA. From left to right severe, mild affected and wild type (WT) embryos at 24 hpf. WT zebrafish show expression in the hindbrain, hindbrain-midbrain boundary, neural tube, mesoderm, optic stalk, otic vesicle, and pronephric duct. Microinjection 62.5 μM mRNA, 125 μM mRNA and 250 μM mRNA resulted in respectively 48%, 71% and 61% malformed zebrafish. There was no correlation between mRNA dosage and severity of malformation.