Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene

Abstract

Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Caucasian and 61 Hispanic patients with non-syndromic lumbar-sacral myelomeningocele. We also performed aCGH analysis on the parents of affected individuals with rare CNVs where parental DNA was available (42 sets). Among the eight de novo CNVs that we identified, three generated copy number changes of entire genes. One large heterozygous deletion removed 27 genes, including PAX3, a known spina bifida-associated gene. A second CNV altered genes (PGPD8, ZC3H6) for which little is known regarding function or expression. A third heterozygous deletion removed GPC5 and part of GPC6, genes encoding glypicans. Glypicans are proteoglycans that modulate the activity of morphogens such as Sonic Hedgehog (SHH) and bone morphogenetic proteins (BMPs), both of which have been implicated in NTDs. Additionally, glypicans function in the planar cell polarity (PCP) pathway, and several PCP genes have been associated with NTDs. Here, we show that GPC5 orthologs are expressed in the neural tube, and that inhibiting their expression in frog and fish embryos results in NTDs. These results implicate GPC5 as a gene required for normal neural tube development.

Figure 1.

Network analysis of coding genes overlapping rare CNVs. Module annotations are done with false discovery rate (FDR) < 0.05. Solid line—annotated interaction; dashed line—predicted interaction; arrow end—activating interaction; straight end—inhibiting interaction. Sources for annotations come from: K, KEGG, P, Panther, R, Reactome, N, NCI Pathways, B, BioCarta, C, CellMap.

Figure 2.

Identification of a de novo heterozygous deletion in a spina bifida proband which removes the polarity genes glypicans GPC5 and GPC6. Segmentation plots of aCGH data generated by Roche NimbleGen software of mother (top), father (middle) and proband (bottom). Genes within this region are indicated below the segmentation plots. Note that the entire GPC5 gene is removed by the deletion, whereas only the first two exons of GPC6 are removed.

Figure 3.

gpc5 morpholinos inhibit neural tube closure in X. tropicalis embryos. (A and B) Tailbud stage X. tropicalis embryos injected with gpc5-MOs (B) or left uninjected (A). (C and D) Stage 22/23 embryos injected with control morpholino (co-MO; C) or 10 ng gpc5-MO (D). Arrow in (D) indicates open neural tube. (E–G) Embryos injected with a co-MO (E) or gpc5-MO (F and G) were stained for pax6 by in situ hybridization. Arrow in (G) indicates open neural tube. Dorsal views, anterior is toward the top in (C, D) and toward the left in (A, B; E–G).

Figure 4.

gpc5 MO disrupt morphogenesis of the trunk in zebrafish embryos. Abnormal morphogenesis of the neural tube and surrounding tissue in gpc5 MO-injected embryos compared with controls, (A–J). Embryos fixed at 14 hpf (A and B) or 24 hpf (C–J) were stained for myod (A–D), a marker of somatic mesoderm; elavl3 (E–H), a marker of differentiated neurons; or ntl (I,J), a marker of the notochord. In comparison to (E) control MO-injected embryos, the hindbrain is broader and neurons appear disorganized in (F) gpc5 MO-injected embryos. This is highlighted in transverse trunk cross sections of (G) control and (H) gpc5 MO-injected embryos. In (A) control MO-injected embryos, somites exhibit a classic chevron shape, while in (B) gpc5 MO-injected embryos, myod expression is compressed. (I and J) In lateral view, the notohord is clear in (I) control MO-injected embryos, while it is absent in (J) gpc5 MO-injected embryos.