Recessive and dominant mutations in retinoic acid receptor beta in cases with microphthalmia and diaphragmatic hernia

Abstract

Anophthalmia and/or microphthalmia, pulmonary hypoplasia, diaphragmatic hernia, and cardiac defects are the main features of PDAC syndrome. Recessive mutations in STRA6, encoding a membrane receptor for the retinol-binding protein, have been identified in some cases with PDAC syndrome, although many cases have remained unexplained. Using whole-exome sequencing, we found that two PDAC-syndrome-affected siblings, but not their unaffected sibling, were compound heterozygous for nonsense (c.355C>T [p.Arg119(∗)]) and frameshift (c.1201_1202insCT [p.Ile403Serfs(∗)15]) mutations in retinoic acid receptor beta (RARB). Transfection studies showed that p.Arg119(∗) and p.Ile403Serfs(∗)15 altered RARB had no transcriptional activity in response to ligands, confirming that the mutations induced a loss of function. We then sequenced RARB in 15 subjects with anophthalmia and/or microphthalmia and at least one other feature of PDAC syndrome. Surprisingly, three unrelated subjects with microphthalmia and diaphragmatic hernia showed de novo missense mutations affecting the same codon; two of the subjects had the c.1159C>T (Arg387Cys) mutation, whereas the other one carried the c.1159C>A (p.Arg387Ser) mutation. We found that compared to the wild-type receptor, p.Arg387Ser and p.Arg387Cys altered RARB induced a 2- to 3-fold increase in transcriptional activity in response to retinoic acid ligands, suggesting a gain-of-function mechanism. Our study thus suggests that both recessive and dominant mutations in RARB cause anophthalmia and/or microphthalmia and diaphragmatic hernia, providing further evidence of the crucial role of the retinoic acid pathway during eye development and organogenesis.

Figure 1

Mutations in RARB in Individuals with PDAC Syndrome Sanger sequencing confirmed segregation of the recessive mutations in RARB in family A (A) and revealed that the mutations were de novo in families B (B), C (C), and D (D).

Figure 2

Localization and Impact of the Mutations in RARB (A) Shown are the positions of the mutations with respect to the different RARB Ensembl-annotated transcripts that are predicted to produce proteins. Numbering on top is based on the cDNA positions of Ensembl ENST00000330688 (identical to RefSeq NM_000965.3). (B) A schematic of RARB shows the DNA-binding and hormone-binding domains. Arrowheads above the protein show the positions of the variants. (C) The HomoloGene-generated amino acid alignment of human RARB and its predicted orthologs shows the conservation of the p.Arg387 residue. (D) The three-dimensional structure of RARB (Protein Data Bank ID 4DM8) in the presence of the RA ligand (purple) shows the proximity of the Arg387 residue in helix 11 to the RA ligand.

Figure 3

Transcriptional Response of Human RARB Variants to RA Ligands HEK293 cells were seeded in 24-well plates and transfected with 100 ng per well of expression plasmid encoding either Gal4 fusion of wild-type human RARB or p.Arg119^∗, p.Ile403Serfs^∗15, p.Arg387Ser, or p.Arg387Cys RARB variants in the presence of 500 ng of UAStkLuc reporter-gene construct. All variant constructs, including p.Ile403Serfs^∗15 (carrying the additional out-of-frame amino acid extension), were generated by site-directed mutagenesis. Cells were treated with 1 μM atRA, 1 μM 9-cis RA, or vehicle (DMSO; 1/1,000, v/v) for 16 hr. Luciferase values were normalized to β-galactosidase activity and expressed as a fold response compared to those of empty Gal4-transfected control cells. Data were derived from three independent experiments performed in triplicate. ^∗p < 0.005 versus wild-type RARB response to the respective RA ligand. Values represent means, and error bars represent SEs.