Mutations in BMP4 cause eye, brain, and digit developmental anomalies: overlap between the BMP4 and hedgehog signaling pathways

Abstract

Developmental ocular malformations, including anophthalmia-microphthalmia (AM), are heterogeneous disorders with frequent sporadic or non-Mendelian inheritance. Recurrent interstitial deletions of 14q22-q23 have been associated with AM, sometimes with poly/syndactyly and hypopituitarism. We identify two further cases of AM (one with associated pituitary anomalies) with a 14q22-q23 deletion. Using a positional candidate gene approach, we analyzed the BMP4 (Bone Morphogenetic Protein-4) gene and identified a frameshift mutation (c.226del2, p.S76fs104X) that segregated with AM, retinal dystrophy, myopia, brain anomalies, and polydactyly in a family and a nonconservative missense mutation (c.278A-->G, p.E93G) in a highly conserved base in another family. MR imaging and tractography in the c.226del2 proband revealed a primary brain developmental disorder affecting thalamostriatal and callosal pathways, also present in the affected grandmother. Using in situ hybridization in human embryos, we demonstrate expression of BMP4 in optic vesicle, developing retina and lens, pituitary region, and digits strongly supporting BMP4 as a causative gene for AM, pituitary, and poly/syndactyly. Because BMP4 interacts with HH signaling genes in animals, we evaluated gene expression in human embryos and demonstrate cotemporal and cospatial expression of BMP4 and HH signaling genes. We also identified four cases, some of whom had retinal dystrophy, with "low-penetrant" mutations in both BMP4 and HH signaling genes: SHH (Sonic Hedgehog) or PTCH1 (Patched). We propose that BMP4 is a major gene for AM and/or retinal dystrophy and brain anomalies and may be a candidate gene for myopia and poly/syndactyly. Our finding of low-penetrant variants in BMP4 and HH signaling partners is suggestive of an interaction between the two pathways in humans.

Figure 1

MLPA Analysis of Cases 1 and 2, BMP4 Mutational Analysis of Cases 3 and 4, and Clinical and Pedigree Data of Case 3 (A) MLPA of case 1 and 2. The figure shows a region of the Genotyper trace that includes the BMP4 and OTX2 peaks plus four control peaks. A deletion of the BMP4 and OTX2 MLPA probe binding site is demonstrated by a 50% reduction in peak height. (B) BMP4 mutation analysis in two families with congenital eye abnormalities. The top row shows a schematic diagram of the human BMP4 cDNA with the positions of the exons and their corresponding translated protein domains. Filled black circles indicate locations of mutations; color-coded vertical arrowheads show the position of each mutation with the corresponding case number. The bottom row shows sequence traces of the affected individuals and their family members (gmo, grandmother; mau, maternal aunt; and bro, brother). (C) Facial views of case 3 and his family members. (C_i) shows case 3 with right anophthalmia (wearing prosthesis), left microanterior segment with coloboma. (C_ii) shows case 3 details of left eye with iris coloboma. (C_iii) shows case 3 left fundus with chorioretinal colobomas, hypoplastic tilted disc. Shown in (C_iv), the facial view of case 3эs grandmother. Shown in (C_v), feet of case 3′s grandmother showing position of right postaxial polydactyly (arrow). In (C_vi) is the facial view of case 3′s mother. (C_vii) shows the pedigree of case 3, as follows: or = myopia; or = anophthalmia, microphthalmia and retinal dystrophy; or = polydactyly; and or = brain anomalies. Shown in (C_viii), the right hand of case 3's grandmother with webbing.

Figure 2

MR Imaging, Diffusion Tractography and Electrodiagnostic Results of Case 3 and Controls MRI structural images (A–F) from case 3 (A and E) sagittal, (B) coronal, (C, D, and F) transaxial views demonstrating thinning of the corpus callosum in (A), and symmetric widening of the lateral ventricles, particularly the trigones in (B) and (C). The hippocampal (arrows, [D]) and sellar structures (E) appear intact. Orbital imaging (F) demonstrates right anophthalmia with a primary orbital implant behind the ocular remnant and slightly enlarged size of the left globe with hypoplasia of anterior structures including iris, lens, and ciliary apparatus. The right optic nerve and chiasm were atrophic, whereas the left optic nerve appeared intact. Thalamo-occipital connectivity scans (G and H) in a representative control subject (G) show symmetrically formed visual pathways (hot colors, for example, red or orange denotes strong evidence of connectivity) and normal structural imaging. In case 3 (H), asymmetry and disruption of the pathways are shown. Diffusion tractography of case 3 and controls (I and J), which assesses connectivity from a manually defined callosal mask to predefined cortical targets, namely prefrontal (PFC), premotor (PMC), primary motor (M1) and somatosensory (S1), occipital (OCC), temporal (TMP), and parietal cortex (PPC), shows reduced interhemispheric connectivity especially relating to M1 and S1 in case 3, whereas connectivity for OCC, TMP, and PPC appears relatively spared. Thalamocortical connectivity score in healthy controls = 1.7 ±0.48 (mean ± SD); case 3 = 4.34; Z = 5.47, p < 0.01). Visual evoked potentials and pattern ERGs (K) and full-field ERGs (L) from the left eye (LE) of case 3 are compared with representative normal examples. Full-field ERGs show moderate-to-severe amplitude reduction and peak-time delay, consistent with generalized retinal dysfunction affecting both rods and cones. Pattern ERG P50 component reduction is in keeping with severe macular involvement. Pattern and flash VEPs are relatively well preserved. LU indicates log units greater (+) or less (−) than the ISCEV standard flash.

Figure 3

Expression Studies of BMP4, SHH, PTCH1, GLI1, and GLI3 in the Developing Human Eye Eye formation in the human embryo at CS 13 (A and B), 14 (C), 17 (D–H), 20 (I–K), and 21 (L–N). Coronal sections through the eye were analyzed by in situ hybridization for the expression for BMP4 (A–D), SHH (E, J, and M), PTCH1 (F, K, and N), GLI1 (G), and GLI3 (H). The following abbreviations are used: El, eye lid; Lf, lens fiber; Rs, retina space; Nl, neural layer of optic cup; Ol, outer layer of optic cup; Nr, neural retina; Rpe, retinal pigment epithelium; Le, lens; Ce, corneal ectoderm; On, optic nerve; Os, optic stalk; Cse, corneal epithelium; and Dl, developing lens. The scale bar for (A)–(H) represents 1000 μm and that for (I)–(N) 1000 μm. Arrows indicate areas of strong expression.

Figure 4

Expression Studies of BMP4, SHH, PTCH1, GLI1, and GLI3 in the Developing Human Brain Coronal sections through the diencephalon of CS17 and 18 human embryos were analyzed by in situ hybridization for the expression of BMP4 (A, F, K, N, and Q), SHH (B, G, L, O, and R), PTCH1 (C, H, M, P, and S), GLI1 (D and I), and GLI3 (E and J). The following abbreviations are used: Rp, Rathke's pouch; Ir, Infundibular recess; Lt, lamina terminalis; Mge, medial ganglionic eminence; Df, diencephalic floor; Tv, third ventricle; Fp, floor plate; Dh, developing hypothalamus; Pcp, prechordal plate; Lv, lateral ventricle; and Bl, basal lamina. The scale bar for (A)–(E) represents 1000 μm, for (K)–(M), 1000 μm, for (F)–(J), 200 μm, for (N)–(P), 100 μm, and for (Q)–(R), 50 μm. Arrows indicate areas of strong expression.

Figure 5

Digit Formation in the Human Embryo at CS20 and 23 All sections were cut across the dorsal ventral axis with respect to the hand plate. In situ analysis of mRNA expression for BMP4 (A, D, G, J, M, and P), SHH (B, E, H, K, N, and Q), and PTCH1 (C, F, I, L, O, and R). (A)–(I) show CS20 stages; (J)–(R) show CS23 stages. The following abbreviations are used: Iz, interdigital zone; Di, digit; Te, tendon; Ca, cartilage; Jc, joint capsule; and Per, pericardium; and Dct, dense connective tissue. The scale bar for (A)–(C) and that for (J)–(L) represent 1000 μm, for (D)–(F) and (M)–(O), 500 μm, and for (G)–(I) and (P)–(R), 50 μm. Arrows indicate areas of strong expression.