We collected various biomarkers to investigate how arsenic contributes to the risk of spina bifida. Environmental exposure to arsenic is of particular concern and may be a potential contributor to spina bifida risk, especially in areas of the world with high arsenic exposures and micronutrient deficiencies. Folic acid supplement use reduces the risk of spina bifida , and fortification of staple foods with folic acid has been successful in decreasing spina bifida rates in multiple countries 4, 5.
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Although our study is the first to our knowledge to report an association between a variant in the ASB2 gene and neural tube defect risk, this gene has previously been implicated in early developmental processes, specifically heart defects. This study adds to the limited, but growing, research focusing on comprehensive scans of the genome, rather than examining a small subset of genes, for risk variants for spina bifida. Characteristics of the study participants, including children with spina bifida, controls, and their parents, for each of the statistical models (mean ± SD, except where noted). For family-based association, standard transmission disequilibrium chi-square tests (TDT; –tdt) were performed on trio samples for 370,617 variants. Standard case/control allelic association tests were performed using PLINK software (Purcell et al., 2007) to determine the relationship between genetic variants and spina bifida case status. Five children, two fathers, and three mothers included in at least one of the analysis models of the current study belonged to a case family and were later reassigned to be categorized as control families after surgery on the child revealed an updated diagnosis of lipomeningocele.
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The canonical Wnt/β-catenin pathway, along with the sonic hedgehog and noncanonical WNT/planar cell polarity pathways, are critical in directing cell differentiation and convergent extension of the neural plate during neural tube closure (Wolujewicz & Ross, 2019). A study by Li et al. (Li et al., 2021) observed that LHPP overexpression inhibits the proliferation and invasion of glioblastoma cells and downregulates the activity of Wnt/β-catenin signaling. A study by Métais et al. (Métais et al., 2018) using Asb2 knockout embryos demonstrated that the absence of Asb2a, an isoform of Asb2, can lead to embryonic mortality in mice as a result of heart defects. Genotype frequencies for the significant variants from the trio transmission disequilibrium test model. Visualization of significant single nucleotide polymorphisms from GWAS analysis was done using Manhattan plots (qqman (Turner, 2018) package in R). The effect of amino acid substitutions in the significant variants from the models was predicted using the PolyPhen-2 Web tool (Adzhubei et al., 2013), with a potential prediction outcome of probably damaging, possibly damaging, or benign.
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DNA extraction of saliva samples from children and their parents was performed using the Maxwell® RSC Buccal Swab DNA kit (Promega, Madison, WI, USA), adhering to the manufacturer’s protocol. Saliva samples were shaken for 5 seconds and stored at room temperature until shipped to Boston for long-term storage and DNA extraction. ASeven samples failed the Broad Institute QC due to call rates less than 97% and/or as a result of a sex mismatch. The Bangladesh Medical Research Council and the Human Research Committees at Boston Children’s Hospital and NINS approved all study protocols, and informed consent was obtained from parents prior to enrollment. We included individuals with lipomeningocele, which is characterized by fatty tissue connected to the spinal cord (Agopian et al., 2012), and their families in the control group as this defect is believed to occur at different developmental stages than myelomeningocele and meningocele (Agopian et al., 2012; Chan et al., 2017).
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