Exome analysis identified a novel mutation in the RBP4 gene in a consanguineous pedigree with retinal dystrophy and developmental abnormalities

Abstract

Retinitis Pigmentosa (RP) is a common form of retinal degeneration characterized by photoreceptor degeneration and retinal pigment epithelium (RPE) atrophy causing loss of visual field and acuities. Exome sequencing identified a novel homozygous splice site variant (c.111+1G>A) in the gene encoding retinol binding protein 4 (RBP4). This change segregated with early onset, progressive, and severe autosomal recessive retinitis pigmentosa (arRP) in an eight member consanguineous pedigree of European ancestry. Additionally, one patient exhibited developmental abnormalities including patent ductus arteriosus and chorioretinal and iris colobomas. The second patient developed acne from young age and extending into the 5(th) decade. Both patients had undetectable levels of RBP4 in the serum suggesting that this mutation led to either mRNA or protein instability resulting in a null phenotype. In addition, the patients exhibited severe vitamin A deficiency, and diminished serum retinol levels. Circulating transthyretin levels were normal. This study identifies the RBP4 splice site change as the cause of RP in this pedigree. The presence of developmental abnormalities and severe acne in patients with retinal degeneration may indicate the involvement of genes that regulate vitamin A absorption, transport and metabolism.

Figure 1. Genetic analysis of a family with retinal degeneration.

A. Pedigree of the family with retinal degeneration. Family members that were available for study were genotyped for the RBP4 c.111+1 G>A mutation. B. The reverse complement sequence of the heterozygous subject IV-3 (G/A) and the homozygous subject IV-2 (A/A) are shown. C. Protein encoding transcripts of RBP4 reported by Ensembl (http://uswest.ensembl.org). Three protein coding transcripts are reported by Ensembl. Transcripts ENST00000371464 and ENST00000371467 encode the same protein (CCDS 31249). ENST00000371469 does not have a CCDS identifier. The RBP4 c.111+1 mutation would affect all three of the predicted protein coding transcripts.

Figure 2. Ocular examination of affected patients IV- 2 at age 63 and IV-4 at age 55.

Patient IV-2 exhibited anterior segment dysgenesis with a small cornea, inferiorly displaced pupil, and inferior coloboma of the iris with corneal limbal neovascularization (panel A). The fundus shows an inferior coloboma of the retina and choroid and severe chorioretinal atrophy (panel B). Patient IV-4 has normal anterior segments coloboma or other abnormalities (panel C). The fundus in both eyes was similar with widespread retinal degeneration comprising extensive peripheral retinal atrophy, “bone spicule” pigmentation throughout except for the central-most macula, and markedly attenuated retinal arterioles (panel D).

Figure 3. Goldman Visual Field (GVF) of the patient IV-4 at age 38 and at age 55.

GVF demonstrated progressive loss from retinal disease. At age 38, both eyes showed a central “tunnel” vision of 15 degrees and large peripheral temporal islands OU (panel A and B), but by age 55, these had constricted to less than 5 degrees, with a small temporal islands for both eyes (panel C and D).

Figure 4. Electroretinogram responses from patient IV-4 with photopic single flash and 30 Hz flicker response stimuli.

Both panels show responses at younger age 38 years on top and at 55 years on bottom. The single flash cone responses at age 38 was reduced approximately 80% from normal and was no longer detectable 17 years later (Panel A). The cone flicker response was similarly reduced at age 38 and further diminished by age 55 (Panel B).

Figure 5. Serum RBP4 and transthyretin (TTR) levels in affected and unaffected family members.

Serum RBP4 and TTR levels were examined by western blot analysis as described in Methods. A) The patients, IV-2 and IV-4 had undetectable levels of wild type RBP4 in contrast to their unaffected sibling and the control. A faint band was observed in the region of predicted mutant RBP4 in both patients and their unaffected sibling (Asterisk). B) TTR levels (TTR monomer (15 kDa) and dimer (30 kDa) were indistinguishable between affected and unaffected subjects.