Burden of rare deleterious variants in WNT signaling genes among 511 myelomeningocele patients

Abstract

Genes in the noncanonical WNT signaling pathway controlling planar cell polarity have been linked to the neural tube defect myelomeningocele. We hypothesized that some genes in the WNT signaling network have a higher mutational burden in myelomeningocele subjects than in reference subjects in gnomAD. Exome sequencing data from 511 myelomeningocele subjects was obtained in-house and data from 29,940 ethnically matched subjects was provided by version 2 of the publicly available Genome Aggregation Database. To compare mutational burden, we collapsed rare deleterious variants across each of 523 human WNT signaling genes in case and reference populations. Ten WNT signaling genes were disrupted with a higher mutational burden among Mexican American myelomeningocele subjects compared to reference subjects (Fishers exact test, P ≤ 0.05) and seven different genes were disrupted among individuals of European ancestry compared to reference subjects. Gene ontology enrichment analyses indicate that genes disrupted only in the Mexican American population play a role in planar cell polarity whereas genes identified in both populations are important for the regulation of canonical WNT signaling. In summary, evidence for WNT signaling genes that may contribute to myelomeningocele in humans is presented and discussed.

Fig 1. Variant filtering and annotation workflow.

The analysis pipeline included filtering variants based on sample and variant-level criteria for quality control. The pipeline also included annotating variants with gene names, reference population frequencies, and CADD phred scores so that variant counts could be collapsed over a gene’s coding region and so that the analysis could focus on likely deleterious, rare variants. Abbreviations: VCF = variant call format, GATK = Genome Analysis Toolkit, dbNSFP = Database of Non-synonymous Single-nucleotide Variant Functional Predictions version 4.0b1a, gnomAD = Genome Aggregation Database version 2.

Fig 2. Distribution of affected genes per subject.

This figure compares Mexican-American (MA) myelomeningocele subjects to European ancestry (EA) myelomeningocele subjects by illustrating how many disrupted genes each subject possessed. Green circles represent MA myelomeningocele data and blue squares represent EA myelomeningocele data. (A) The horizontal axis measures the number of genes that are disrupted by qualifying variants. The vertical axis depicts the number of subjects who possess that number of disrupted genes. (B) The horizontal axis counts only the number of genes that indicated nominally significant risk in the mutational burden analysis. The vertical axis shows the number of subjects with that number of disrupted genes. The vertical dotted lines from either graph represent the median number of disrupted genes possessed by subjects, with green lines representing MA and blue representing EA medians. Both populations followed a similar distribution for how many genes are disrupted per subject, complete with a similar median. However, the Mexican Americans tended to have more nominally significant disrupted genes.

Fig 3. Altered WNT trafficking.

The proteins PORCN, SDC1, and CPE are all involved in WNT ligand trafficking. Deleterious variants in PORCN may prevent WNT’s acetylation which is necessary for WNT to leave the endoplasmic reticulum. Loss of PORCN function would stop WNT from leaving the cell. CPE prevents WNT from binding cell surface receptors on the target cell. Loss of CPE function may indirectly increase WNT’s effect on the target cell. Proteoglycans like SDC1 have been implicated as regulators in WNT distribution, though the exact mechanism is not yet known.

Fig 4. Altered planar cell polarity.

DVL2, PRICKLE2, and FUZ genes were each disrupted in the myelomeningocele populations. The FZD-DVL complex is necessary to establish planar cell polarity. DVL proteins are also needed to translocate FUZ to cilia. FUZ is essential for the development of a cilium. PRICKLE inhibits the formation of the FZD-DVL complex.

Fig 5. Altered β-catenin.

A visual summary of the WNT/β-catenin cascade including genes disrupted in the myelomeningocele populations. The level of β-catenin, which is coded for by the human homolog CTNNB1, is regulated by many proteins in the WNT ligand’s target cell. The proteins CSNK1G, DVL2, DDB1, PSMD3, PTPRU, Fermitin 2 (coded by FERMT2), CPE, PPP2R1A (not shown), and SOSTDC1 contribute to β-catenin’s regulation and all have higher mutational burdens in one of the myelomeningocele populations compared to gnomAD references.