High-throughput custom capture sequencing identifies novel mutations in coloboma-associated genes: Mutation in DNA-binding domain of retinoic acid receptor beta affects nuclear localization causing ocular coloboma

Abstract

Uveal coloboma is a potentially blinding congenital ocular malformation caused by the failure of optic fissure closure during the fifth week of human gestation. We performed custom capture high-throughput screening of 38 known coloboma-associated genes in 66 families. Suspected causative novel variants were identified in TFAP2A and CHD7, as well as two previously reported variants of uncertain significance in RARB and BMP7. The variant in RARB, unlike previously reported disease mutations in the ligand-binding domain, was a missense change in the highly conserved DNA-binding domain predicted to affect the protein's DNA-binding ability. In vitro studies revealed lower steady-state protein levels, reduced transcriptional activity, and incomplete nuclear localization of the mutant RARB protein compared with wild-type. Zebrafish studies showed that human RARB messenger RNA partially reduced the ocular phenotype caused by morpholino knockdown of rarga gene, a zebrafish homolog of human RARB. Our study indicates that sequence alterations in known coloboma genes account for a small percentage of coloboma cases and that mutations in the RARB DNA-binding domain could result in human disease.

Keywords: coloboma; custom capture; high-throughput sequencing; retinoic acid receptor beta.

Figure 1

Clinical findings in four coloboma families. Family 1: The proband (1.1) had bilateral coloboma in the retina and iris coloboma in the left eye; the mother (1.2) presented with left eye microcornea and posterior coloboma and right eye with chorioretinal coloboma inferior to the optic disc. Family 2: The proband (2.1) had left eye microcornea and coloboma in the optic nerve and inferior retina. Family 3: The proband (3.1) had bilateral coloboma. Family 4: The proband (4.1) had bilateral chorioretinal coloboma; the right eye was microphthalmic. OD, right eye; OS, left eye

Figure 2

Arg144Gln mutation in RARB affects the binding ability of the DNA‐binding domain. (a) RARB protein with six domains and the approximate location of the Arg144Gln mutation in the DNA‐binding domain. (b) Conservation of the mutated amino acid in the DNA‐binding domain across vertebrate species. (c) Molecular modeling of the interaction of the DNA‐binding domain (orange) with the DNA (beige). The rest of the RARB protein is shown in light blue. (d) Structural superimposition of the two DNA‐binding domains from wild‐type protein (orange) and Arg144Gln mutant variant (magenta) shown in the same DNA double helix groove. In the Arg144Gln mutant, the orientation of the α‐helix interacting with the DNA (green arrowhead) is altered. Location of Arg137 indicated by white arrowhead

Figure 3

Arg144Gln mutation in RARB results in reduced steady‐state exogenous protein levels, retention in the cytoplasm and affects transcriptional activity. (a) Western blot showing reduced DYK‐tagged RARB‐mutant compared with RARB wild‐type protein in HEK 293‐transfected cells treated with retinoic acid (RA). GFP was used as a control for transfection efficiency and ACTB as a loading control. DYK‐tagged constructs were used for transfection and proteins were detected by DYK, GFP, and ACTB antibodies. (a′) Densitometric analysis of the RARB–DYK bands with normalization to the ACTB. (b–e) Immunofluorescence in HEK 293 cells transfected with wild‐type or mutant RARB–GFP expression constructs followed by RA treatment. Both RARB–GFP and RARB‐mutant‐GFP proteins localized to the cytoplasm in the absence of RA. Upon treatment with RA, wild‐type RARB protein localized to the nucleus, while RARB‐mutant protein was mostly retained in the cytoplasm as revealed by the GFP tag. Scale bar in (e) applies to all images. (b′) Bar graph of the percentage of cells showing predominant localization of the transfected protein in the cytoplasm or the nucleus. Values on the graph indicate the total number of cells counted. Only immunolabeled cells were counted from five different fields. Cells with predominant localization in the cytoplasm (brown); cells with predominant localization in the nucleus (blue). With RA treatment, RARB wild‐type protein showed significantly more nuclear localization compared with the RARB‐mutant protein (p < .01). (f) Nuclear and cytoplasmic fractions separated from transfected HEK 293 cells treated with RA. Wild‐type RARB protein was mostly localized to the nucleus, while RARB‐mutant protein was observed both in the nucleus and in the cytoplasm. (f′) Percentage expression in the nucleus and cytoplasm of RARB–DYK bands from the densitometric analysis. The mutant protein showed reduced expression and localization to the nucleus compared with the wild‐type protein. (g) Protein degradation assay. A T ₀ timepoint was harvested before cycloheximide addition (0 hr). The mutant protein was barely visible after 12 hr of cycloheximide treatment, whereas, wild‐type RARB protein was still detected. The red arrow indicates the RARB–DYK band used for densitometric analysis. (g′) Densitometric analysis of the RARB–DYK bands normalized to ACTB. (h) qRT‐PCR of wild‐type and mutant RARB transcripts from the transfected HEK 293 cells normalized to GAPDH and to the neomycin‐resistance gene from the expression vector construct. Expression of the RARB‐mutant transcript was significantly reduced compared with the wild‐type transcript. ***p < .01. (i) Transcriptional activity of RARB wild‐type and mutant proteins. Transcriptional activity is expressed as Firefly/Renilla luciferase activity ratio. RA treatment induced a significant increase in the transcriptional activity of wild‐type RARB but not of RARB‐mutant, ***p < .001. GFP, green fluorescent protein; HEK 293, human embryonic kidney 293; qRT‐PCR, quantitative real‐time polymerase chain reaction

Figure 4

RARB‐mutant protein does not alter the localization of RXR proteins. (a–p) Immunofluorescence of HEK 293 cells treated with DMSO/RA following transfection with GFP‐tagged wild‐type RARB or RARB‐mutant and RFP‐tagged RXRA expression constructs. Wild‐type RARB along with RXRA protein appeared to translocate to the nucleus upon RA treatment whereas partial retention of RXRA protein was seen in the cytoplasm along with the RARB‐mutant protein as revealed by colocalization of GFP and RFP signals from the fusion proteins. Scale bar in (p) applies to all images. (q) Western blots of protein lysates from cytoplasmic and nuclear fractions separated from cells cotransfected with wild‐type or mutant RARB–DYK and RXRA–RFP expression constructs and treated with RA. RXRA localization appeared not affected in cells cotransfected either with RARB wild‐type or mutant expression constructs. However, RARB wild‐type protein was mostly localized to the nucleus while the RARB‐mutant protein was mostly localized to the cytoplasm. (q′) Expression percentage of the RARB–DYK and RXRA–RFP bands from the densitometric analysis. The mutant RARB protein showed reduced expression and translocation to the nucleus compared with the wild‐type protein while the RXRA showed no difference in localization. DMSO, dimethyl sulfoxide; GFP, green fluorescent protein; HEK 293, human embryonic kidney 293; RA, retinoic acid

Figure 5

Ocular coloboma phenotype and partial rescue in rarga morpholino (MO) knockdown zebrafish embryos. (a) rarga MO‐injected embryos show a slightly smaller body and head sizes compared with uninjected zebrafish embryos at 48 hr postfertilization (hpf). (b) Uninjected wild‐type with normal eye. (c) Standard control MO‐injected embryos. Ocular size is comparable to the wild‐type uninjected embryos and both reveal no coloboma in the eye or other phenotypes at 3.75–7.5 ng MO concentrations. (d–e) rarga MO‐injected embryos revealed concentration‐dependent ocular coloboma as Grade 2 (narrow, d) or Grade 3 (wide, e) optic fissure closure defects. The red bars indicate the margins of the optic fissure. Scale bar in (e) applies to panels b–e. (f) rarga transcript expression by qRT‐PCR was significantly reduced in MO knockdown zebrafish embryos; ***p < .001. (g) Human RARB mRNA but not RARB‐mutant mRNA partially reduced the coloboma phenotype in rarga MO knockdown embryos. Embryos were injected with rarga splice‐blocking MO along with either human wild‐type RARB or RARB‐mutant mRNA and coloboma phenotype was analyzed after 48 hpf. The values on the bars represent the number of embryos injected or analyzed. qRT‐PCR, quantitative real‐time polymerase chain reaction; mRNA, messenger RNA