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. 2009 Dec;126(6):791-803.
doi: 10.1007/s00439-009-0730-x.

Reduced TFAP2A function causes variable optic fissure closure and retinal defects and sensitizes eye development to mutations in other morphogenetic regulators

Gaia Gestri  1 , Robert J Osborne, Alexander W Wyatt, Dianne Gerrelli, Susan Gribble, Helen Stewart, Alan Fryer, David J Bunyan, Katrina Prescott, J Richard O Collin, Tomas Fitzgerald, David Robinson, Nigel P Carter, Stephen W Wilson, Nicola K Ragge
Affiliations

Reduced TFAP2A function causes variable optic fissure closure and retinal defects and sensitizes eye development to mutations in other morphogenetic regulators

Gaia Gestri et al. Hum Genet. 2009 Dec.

Abstract

Mutations in the transcription factor encoding TFAP2A gene underlie branchio-oculo-facial syndrome (BOFS), a rare dominant disorder characterized by distinctive craniofacial, ocular, ectodermal and renal anomalies. To elucidate the range of ocular phenotypes caused by mutations in TFAP2A, we took three approaches. First, we screened a cohort of 37 highly selected individuals with severe ocular anomalies plus variable defects associated with BOFS for mutations or deletions in TFAP2A. We identified one individual with a de novo TFAP2A four amino acid deletion, a second individual with two non-synonymous variations in an alternative splice isoform TFAP2A2, and a sibling-pair with a paternally inherited whole gene deletion with variable phenotypic expression. Second, we determined that TFAP2A is expressed in the lens, neural retina, nasal process, and epithelial lining of the oral cavity and palatal shelves of human and mouse embryos--sites consistent with the phenotype observed in patients with BOFS. Third, we used zebrafish to examine how partial abrogation of the fish ortholog of TFAP2A affects the penetrance and expressivity of ocular phenotypes due to mutations in genes encoding bmp4 or tcf7l1a. In both cases, we observed synthetic, enhanced ocular phenotypes including coloboma and anophthalmia when tfap2a is knocked down in embryos with bmp4 or tcf7l1a mutations. These results reveal that mutations in TFAP2A are associated with a wide range of eye phenotypes and that hypomorphic tfap2a mutations can increase the risk of developmental defects arising from mutations at other loci.

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Figures

Fig. 1
Fig. 1
Results of molecular analysis of TFAP2A showing gene and protein structure, MLPA and custom array CGH. a Schematic representation of TFAP2A mRNA showing exon breakdown and number of bases encoded by each exon; splice indicators above depict TFAP2A full-length mRNA (NM003220.2 isoform 1a), while those below describe exon 5 alternatively spliced in the TFAP2A2 isoform (M61156); TFAP2A protein representation showing full-length protein (upper) and M61156 isoform (lower) showing domains. PG proline/glutamine rich domain (for transactivation), B basic domain of DNA binding region, HSH helix-span-helix domain (mediates dimerization and site specific DNA binding). Small arrows show previously reported mutations, large arrows show our cases. b Chromatograms from Cases 1 and 2 and normal controls showing position of variations (arrows). Comparative amino acid sequence alignment of vertebrate TFAP2A and genomic sequence alignment of TFAP2A2 encompassing the regions altered in Cases 1 and 2. c MPLA traces for Cases 3, 4, and 5 showing a 50% reduction in peak height for exons 2, 4, 5, 6 and 7 of TFAP2A compared to maternal and control peaks (no probes were available for exons 1 and 3). d High resolution comparative genomic hybridization (CGH) microarray analysis of the TFAP2A region. The data show an approximate 70 kb deletion in (i) Case 3, (ii) Case 4, and (iii) Case 5 (father), but not in (iv) mother. The position of the TFAP2A gene is shown in green and C6orf218 in blue. Oligonucleotides are colour coded in order of increasing reliability according to their Agilent probe score (where <0.4 is poor and>0.4 is good,>0.8 is very good): 0–0.2 red, 0.2–0.4 yellow, 0.4–0.6 green, 0.6–0.8 blue, 0.8–1.0 black. Chromosome co-ordinates are according to NCBI36
Fig. 2
Fig. 2
Photographs of external features of Cases 1, 3, 4, and 5. Case 1 showing a full face with frontal bossing, R anophthalmia (wearing prosthesis), prominent philtrum; b left iris coloboma c right periorbital subcutaneous lump (presumed dermoid); d right and left external ears showing malformed pinnae; and e close-up of oral cavity demonstrating abnormal dentition (three inferior incisors) and prominent philtrum (f) view of chest showing widely spaced nipples. Case 3: g Full face in early life showing bilateral orbital cysts; h full face showing patient wearing bilateral prosthetic eyes and prominent philtrum; i close-up of microphthalmic remnants; j close-up of oral cavity demonstrating abnormal dentition; k right and left cervical skin defects; l pits in posterior pinna. Case 4: m Full face showing bilateral microphthalmia with R esotropia and hypertropia and inferior orbital cyst; n close of right and left microphthalmic eyes; o feet showing bilateral 2/3 partial syndactyly; p showing ears with mild malformed pinnae; q mild webbing of fingers. Case 5 showing r full face with prominent philtrum; s bilateral 2/3 partial syndactyly feet; t bilateral malformed pinnae
Fig. 3
Fig. 3
In situ hybridization of TFAP2A in mouse and human developing embryos showing expression of TFAP2A. a Sagittal section of 12.5 dpc mouse showing expression of Tfap2a in the nasal process, palate, cerebellum, thalamus, and spinal cord. b Coronal section of CS 15 human eye showing expression of TFAP2A in the anterior lens epithelium. c Coronal section of CS 18 human eye showing expression of TFAP2A in the anterior lens epithelium, and ganglion cell layer of the neural retina. d Coronal section of CS 22 human eye showing expression of TFAP2A in the equatorial region of the lens epithelium, throughout the secondary lens fibres and ganglion cell layer of the neural retina. np nasal process, ce cerebellum, pa palate, th thalamus, sc spinal cord, nr neural retina, le lens
Fig. 4
Fig. 4
Zebrafish studies demonstrating eye and craniofacial defects in tfap2a morpholino-injected larvae and enhanced phenotypes in Wnt and Bmp morphants. a Abrogation of tfap2a function leads to eye and craniofacial defects. (i, ii) Wild-type and tfap2a morpholino-injected larvae at 3.5 dpf; arrow in (ii) points to the expansion of the retinal pigmented epithelium (RPE) towards the midline (black line) due to ventral eye coloboma. (iii, iv) Pharyngeal cartilages from 3.5 dpf wild-type (iii) and tfap2a morpholino-injected larvae (iv), stained with Alcian Blue and shown in ventral view. Palatoquadrate (roof of mouth) and ceratohyal (part of the hyoid bone) (iv, arrows) are severely disorganised in the morphant. Higher magnification of 3.5 dpf embryos showing wild-type (v) and morphant eyes with ectopic RPE (vi) and coloboma (arrow in vii). (viii) Table showing the percentage of the abnormal phenotypes in the tfap2a morpholinoinjected larvae (ectopic RPE, coloboma, small eye, jaw defects). m Meckel, pq palatoquadrate, ch ceratohyal. b Synthetic and enhanced coloboma and anophthalmia phenotypes are revealed by combined abrogation of tfap2a and tcf7l1a. Lateral view of 3.5 dpf larva injected with a low concentration of tfap2a morpholino that does not give rise to any phenotypes in a wild-type background (i), gives rise to a coloboma phenotype in tcf7l1am881/+ heterozygotes (ii) and anophthalmia in the tcf7l1am881/m881 homozygote (iii). PCR-based confirmation of predicted genotypes based upon phenotypes (iv). PCR products were amplified from individual 3.5 dpf larvae with a wild-type phenotype as in (i) (lanes 1–4 16/16 embryos are homozygous for the wild-type allele), a mild phenotype as in (ii) (lane 5–12; 32/36 embryos are heterozygous for the mutant allele as shown in lane 5, 7, 9–12 and 4/36 are homozygous for the mutant allele as in lanes 6 and 8) and an anophthalmia phenotype as in (iii) (lane 13–16; 16/16 embryos are homozygous for the mutant allele). c Synthetic and enhanced eye phenotypes are revealed by combined abrogation of tfap2a and bmp4. Lateral views of 3.5 dpf larvae injected with a low concentration of tfap2a morpholino that does not give rise to any phenotypes in a wild-type background. In 60% of the bmp4st72/+ heterozygotes (i) there is no phenotype (wild-type and heterozygote embryos are indiscernible), whereas in the bmp4st72/st72 homozygotes (ii) there is a prominent coloboma (black brackets). PCR-based confirmation of predicted genotypes based upon phenotypes (iii). PCR products were amplified from individual 3.5 dpf larvae with a wild-type phenotype as in i (lanes 1–4), both wild type (lanes 1 and 4 and heterozygote genotype are present (lanes 2 and 3), and a coloboma phenotype as in ii (lane 5–14). Most affected individuals are bmp4st72/st72 homozygotes although 2 (lanes 5 and 7) are bmp4st72 heterozygotes. The differences in intensity of the PCR bands can result from a difference in the efficiency of the genomic DNA extraction or PCR reaction, or, less likely, from partial DNA digestion
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