Whole-exome sequencing links a variant in DHDDS to retinitis pigmentosa

Abstract

Increasingly, mutations in genes causing Mendelian disease will be supported by individual and small families only; however, exome sequencing studies have thus far focused on syndromic phenotypes characterized by low locus heterogeneity. In contrast, retinitis pigmentosa (RP) is caused by >50 known genes, which still explain only half of the clinical cases. In a single, one-generation, nonsyndromic RP family, we have identified a gene, dehydrodolichol diphosphate synthase (DHDDS), demonstrating the power of combining whole-exome sequencing with rapid in vivo studies. DHDDS is a highly conserved essential enzyme for dolichol synthesis, permitting global N-linked glycosylation. Zebrafish studies showed virtually identical photoreceptor defects as observed with N-linked glycosylation-interfering mutations in the light-sensing protein rhodopsin. The identified Lys42Glu variant likely arose from an ancestral founder, because eight of the nine identified alleles in 27,174 control chromosomes were of confirmed Ashkenazi Jewish ethnicity. These findings demonstrate the power of exome sequencing linked to functional studies when faced with challenging study designs and, importantly, link RP to the pathways of N-linked glycosylation, which promise new avenues for therapeutic interventions.

Figure 1

DHDDS Harbors a Mutation Causing RP (A) Pedigree of the studied family with three affected offspring indicating a recessive trait. (B) Three-dimensional structural model of DHDDS indicates an important role for Arg38 in the organization of the enzyme active site. The Lys42Glu mutant allele, being negatively charged, will likely ion pair with the positively charged Arg38 and, in so doing, may lead to distortion of the active site and compromise the binding of farnesyl-pyrophosphate (arrow). A detailed description of the mechanism is provided in Figure S2. (C) Sanger sequencing confirmed the identified mutation and revealed complete cosegregation with the phenotype. (D) Lys42 (red box) is a highly conserved residue in multiple species and is located close to the active site of the protein (orange box), indicating strong evolutionary constraints.

Figure 2

Photoreceptor Degeneration Induced by Suppression of DHDDS Expression and Illustration of the Involved Pathways of N-Linked Glycosylation (A–F) Microphotographs in (A) and (D) show representative retinal sections of control and fish treated with MO. The areas in red rectangles are shown at higher magnification in (B) and (E). The layers of the retina are indicated by the white bars in (A), including the retinal pigment epithelium (1), the outer nuclear layer (2), the inner nuclear layer (3), the inner plexiform layer (4), and the retinal ganglion cell layer (5). In the retinas treated with MO, the outer segments of photoreceptors are very short, or completely missing (E, arrowhead), compared to the controls (B, arrowhead). Staining of cone outer segments with peanut agglutinin (PNA) highlights that cone outer segments in MO-treated zebrafish (F, arrowhead), but not in controls (C, arrowhead), are missing. Semithin plastic sections (A, B, D, and E) were stained with toluidine blue. Cryosections (C and F) were stained with PNA (conjugated with Alexa Fluor 488). Scale bars represent 25 μm for (A) and (D), 10 μm for (B) and (E), and 20 μm for (C) and (F). (G) A simplified schematic of essential N-linked glycosylation pathways (steps 1–4). An emphasis is given to key processes upstream and downstream of dolichol-PP synthesis (DHDDS) that are also related to RP phenotypes. (1) The cytoplasmic mevalonate pathway produces isoprenoide units, which are (2) chained to ER membrane-bound dolichol species, dolichol-pyrophosphate (dolichol-PP). (3) Dolichol-PP serves as an intermediate lipid to bind a common core of carbohydrates, en bloc, forming the lipid-linked oligosaccharide (LLO). The LLO is “flipped” within the ER membrane to face the ER lumen. (4) Finally, the common core of carbohydrates is transferred to proteins, such as rhodopsin. Along this pathway, mevalonate kinase deficiency is associated with RP, mutations in PMM2 (phosphomannomutase 2; MIM 601785) lead to congenital disorder of glycosylation Ia (CDGIa), often involving RP, and, finally, glycosylation defects in rhodopsin cause RP. N denotes asparagine.