Homozygosity mapping reveals PDE6C mutations in patients with early-onset cone photoreceptor disorders

Abstract

Cone photoreceptor disorders form a clinical spectrum of diseases that include progressive cone dystrophy (CD) and complete and incomplete achromatopsia (ACHM). The underlying disease mechanisms of autosomal recessive (ar)CD are largely unknown. Our aim was to identify causative genes for these disorders by genome-wide homozygosity mapping. We investigated 75 ACHM, 97 arCD, and 20 early-onset arCD probands and excluded the involvement of known genes for ACHM and arCD. Subsequently, we performed high-resolution SNP analysis and identified large homozygous regions spanning the PDE6C gene in one sibling pair with early-onset arCD and one sibling pair with incomplete ACHM. The PDE6C gene encodes the cone alpha subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase, which converts cGMP to 5'-GMP, and thereby plays an essential role in cone phototransduction. Sequence analysis of the coding region of PDE6C revealed homozygous missense mutations (p.R29W, p.Y323N) in both sibling pairs. Sequence analysis of 104 probands with arCD and 10 probands with ACHM revealed compound heterozygous PDE6C mutations in three complete ACHM patients from two families. One patient had a frameshift mutation and a splice defect; the other two had a splice defect and a missense variant (p.M455V). Cross-sectional retinal imaging via optical coherence tomography revealed a more pronounced absence of cone photoreceptors in patients with ACHM compared to patients with early-onset arCD. Our findings identify PDE6C as a gene for cone photoreceptor disorders and show that arCD and ACHM constitute genetically and clinically overlapping phenotypes.

Figure 1

Molecular Genetic Characterization of the PDE6C Gene in Four Families with Autosomal Recessive Cone Photoreceptor Disorders (A) The 10q22-q25 region and the homozygous regions identified in patients of families A and B. The SNPs flanking the homozygous regions and their genomic positions, from the March 2006 UCSC genome build (hg18), are indicated. (B) Protein and genomic structure of PDE6C. GAF, acronym derived from proteins in which these domains were initially identified (cGMP-regulated mammalian phosphodiesterases, cyanobacterial adenylyl cyclases, and a formate-hydrogen lyase transcriptional activator); HDc, domain with highly conserved histidines (H) and aspartic acids (D); cc, coiled-coil domain. The six mutations are labeled M1 through M6. (C) Chromatograms showing the PDE6C mutations. (D) Pedigrees of early-onset cone dystrophy (family A), incomplete achromatopsia (family B), and complete achromatopsia (families C and D) and segregation analysis of the respective PDE6C variants. The parents in family A are not related to each other in the last three generations. BI-1 and BI-2 are first cousins. (E) Evolutionary conservation of the altered amino acid residues in three families. PDE6A and PDE6C are rod and cone α subunits respectively; PDE6B is the rod β subunit. Using the Swiss-Prot database, we searched for their orthologs in as many species as possible and included, with the exception of Danio rerio, those present in all species. White lettered residues on a black background are fully conserved between different species in all three subunits. White lettered residues on a gray background are conserved in most sequences. Black lettered residues with light gray background are similar to amino acid residues in other orthologs and homologs. The arginine residue at position 29 is fully conserved in all orthologs of both α subunits (PDE6C and PDE6A), except for bovine and canine PDE6C. The aspartic acid residue in the GAF-A domain at position 211 is conserved or replaced by a functionally conserved glutamic acid residue in 19 of 20 orthologs and homologs. This observation strongly suggests that the predicted p.E211D substitution in family D is neutral and that the effect of c.633G→C on the splice site is pathologic. The tyrosine residue located in the GAF-B domain at position 323 is completely conserved among all three PDE6 subunits. The methionine residue at position 455 is conserved or substituted by a functionally conserved isoleucine in all PDE6 molecules.

Figure 2

Molecular Modeling of Y323 in the GAF-B Domain of PDE6C The individual side-chain orientations of D322 and Y323 are shown in magenta. A peptide backbone trace of the remainder of the model is shown, with β sheets colored orange and α helices colored blue. The mutation site Y323 is located adjacent to D322. The latter amino acid is shown interacting with bound cGMP via two hydrogen bonds, one originating from the peptide main chain and one originating from the aspartic acid side chain. Hydrogen bonds are indicated by yellow dashed lines.

Figure 3

Electroretinogram of Patient CII-1 An electroretinogram performed with an abbreviated standard International Society for Clinical Electrophysiology of Vision protocol at the age of 4 years without sedation is shown at top, with normal control traces at bottom for purposes of comparison. Note normal rod-specific response (top trace at left), with no significant contribution of cones to combined rod-cone response (bottom trace at left for patient CII-1); dark-adapted oscillatory responses as seen on ascending limb of the b-wave are residual at most. The absence of cone-specific function was demonstrated by the absence of cone-specific response to 30 Hz flicker stimulation (at right).

Figure 4

Retinal Phenotypes of Early-Onset Cone Dystrophy and Complete Achromatopsia in Families A and D (A) Fundus photography of the right eye of patient AII-2, performed at age 51 years, showing myopic changes in the peripapillary region. In the macular region, mild pigmentary changes are present. The arrow denotes the position of the optical coherence tomography (OCT) image in (B). (B) The Heidelberg Spectralis OCT of this patient reveals a serous detachment of the photoreceptor layer in the central fovea. The retinal pigment epithelium is intact, but the outer and inner segments of the photoreceptors are absent. The length of the lesion is ∼500 μm. The OCT cross-section is not fully perpendicular. (C) Fundus photography of the left eye of patient DII-1, performed at age 37 years, showing mild pigmentary changes in the macula. The arrow denotes the position of the OCT image in (D). (D) The Heidelberg Spectralis OCT of this patient displays a large area of ∼1300 μm with absent cone photoreceptors, which involves almost the entire fovea.