PDE6C: Novel Mutations, Atypical Phenotype, and Differences Among Children and Adults

Abstract

Purpose: Genetic variation in PDE6C is associated with achromatopsia and cone dystrophy, with only a few reports of cone–rod dystrophy in the literature. We describe two pediatric and two adult patients with PDE6C related cone and cone–rod dystrophy and the first longitudinal data of a pediatric patient with PDE6C-related cone dystrophy.

Methods: This cohort of four patients underwent comprehensive ophthalmologic evaluation at the National Eye Institute's Ophthalmic Genetics clinic, including visual field testing, retinal imaging and electroretinogram (ERG). Next-generation sequencing–based genetic testing was performed and subsequent analysis of the variants was done through three-dimensional protein models generated by Phyre2 and Chimera.

Results: All cases shared decreased best-corrected visual acuity and poor color discrimination. Three of the four patients had a cone–rod dystrophy, presenting with an ERG showing decreased amplitude on both photopic and scotopic waveforms and a mild to moderately constricted visual field. One of the children was diagnosed with cone dystrophy, having a preserved peripheral field. The children had none to minor structural retinal changes, whereas the adults had clear macular dystrophy.

Conclusions: PDE6C-related cone–rod dystrophy consists of a severe phenotype characterized by early-onset nystagmus, decreased best-corrected visual acuity, poor color discrimination, progressive constriction of the visual field, and night blindness. Our work contributes with valuable information toward understanding the visual prognosis and allelic heterogeneity of PDE6C-related cone and cone–rod dystrophy.

Phosphodiesterase 6 (PDE6) corresponds with 1 of 21 human family members of 11 subfamilies responsible for regulating the intracellular concentration of cyclic nucleotides in every tissue.^1,2 PDE6 is an essential component of the phototransduction cascade, which converts light to electrical signals within the neural retina.³ When light activates this enzyme, intracellular cGMP is hydrolyzed, leading to a change in membrane potential and propagation of the visual cycle.⁴ Rods have a PDE6 catalytic core composed of a heterodimer of PDE6A and PDE6B subunits (inhibited by PDE6G), whereas cones have a catalytic homodimer of PDE6C (inhibited by PDE6H subunits).⁵ This explains why mutations in PDE6A (OMIM #180071), PDE6B (OMIM #180072) and PDE6G (OMIM #180073) cause autosomal recessive retinitis pigmentosa and autosomal dominant congenital stationary night blindness (both rod-related diseases),^6,7 whereas homozygous or compound heterozygous mutations in PDE6C and PDE6H (OMIM #600827 and #601190) lead to predominantly cone dysfunction disorders, such as achromatopsia (ACHM) and autosomal recessive cone dystrophy.^8,9 

Cone dystrophies are characterized by nystagmus, decreased best-corrected visual acuity (BCVA) and poor color vision. Among this group, ACHM is also described as congenital, and largely stationary lack of color discrimination.¹⁰ About 73% (n = 50) of the PDE6C reported mutations (n = 68)^8,11–32 are associated with cone dystrophy (n = 03) and ACHM (n = 47), accounting for 1.0% to 2.4% of all ACHM cases.^13,33 An atypical phenotype reported in recent years with fewer PDE6C mutations (approximately 15%; n = 10)^13–17 is cone–rod dystrophy. This diagnosis not only implies decreased BCVA and color discrimination, but also progressive visual field constriction, night blindness, and decreased scotopic responses in the ERG.³⁴ PDE6C has been screened in cone–rod dystrophy cohorts and has been attributed to up to 1.8% of cases.^16,35,36 The remaining 12% (n = 8) of the mutations were reported to be associated with nonspecified retinal dystrophies (n = 5)^{17,19,22,26,31} and nonretinal diseases (n = 3).^37–39 

In the present series, we report four unrelated individuals with mutations in PDE6C. The two adults and one of the children presented with cone–rod dystrophy, ascertained by ERG and visual field. 

Methods

Clinical Evaluation

Patients were evaluated at the National Eye Institute, National Institutes of Health (Bethesda, MD). They were consented to an National Eye Institute–sponsored, institutional review board–approved clinical research protocol for studying the genetics of inherited eye diseases. All study protocols adhered to the tenets of the Declaration of Helsinki and complied with the Health Insurance Portability and Accountability Act. Four unrelated cases that came for evaluation owing to decreased visual acuity, nystagmus, and poor color discrimination, were found to share the same affected gene (PDE6C) and similar ocular phenotype. 

A complete ophthalmic evaluation was performed, including measurement of BCVA, motility and alignment examination, slit lamp, and dilated fundus evaluation. Additional testing included color, red-free, and autofluorescence retinal imaging (Optos ultrawide-field retinal imaging device, Dunfermline, Scotland), Cirrus optical coherence tomography (OCT; Carl Zeiss Meditec AG, Dublin, CA), and manual Goldmann kinetic perimetry (Goldmann perimeter, Haag-Streit AG, Koniz, Switzerland). The Low Vision Cambridge Color Testing device (LvCCT; Cambridge Research Systems Ltd., Cambridge, UK) was used to test color discrimination. 

ERG was performed in patients who could cooperate with testing. The International Society for Clinical Electrophysiology of Vision standard full-field ERGs were recorded using a commercial electrophysiology system (LKC, Gaithersburg, MD).^40,41 The amplitude and implicit time of both scotopic (dark adapted, 0.01; dark adapted, 3.0; and dark adapted, 10.0) and photopic (light adapted 3.0 and 30 Hz cone flicker) responses were recorded and compared with reference values to evaluate rod and cone function. A summary of the clinical characteristics of the probands affected with PDE6C variants can be found in Table 1. 

Table 1.

 

Clinical Characteristics of Probands Affected With PDE6C Variants

 

Molecular Genetics Study

Genomic DNA was extracted from each patient's blood and different next-generation sequencing–based genetic diagnostic tests (including Sanger confirmation and segregation analysis) were performed at the CLIA-certified laboratory Blueprint Genetics. Patients A and B were tested for mutations in the achromatopsia panel, consisting of six genes (ATF6, CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H). Patients C and D underwent next-generation sequencing–based retinal dystrophy panel testing, which involved 325 genes, including those in the achromatopsia panel. Pathogenic and likely pathogenic variants were further confirmed by bidirectional Sanger sequencing. Copy number variations (deletions and duplications) were also screened in patients C and D using a quantitative PCR assay. After Sanger validation and segregation analysis when parental samples were available, the American College of Medical Genetics guidelines were used to classify the variants for pathogenicity.⁴² In silico predictions were obtained from VarSome (https://varsome.com/).⁴³ Three-dimensional protein models were generated by Phyre2 and Chimera was used to visualize the structure. Clustal omega was used to make multiple sequence alignment of PDE6C orthologous proteins. 

Results

Ophthalmic Findings

Patient A is a 9-year-old Latina girl who was referred for evaluation of decreased visual acuity and poor color discrimination. These symptoms were present and stable since early childhood, per report. Her BCVA was 20/200 OD and 20/250 OS, with correction of mild myopia. Motility examination showed alternating exotropia and low amplitude, moderate frequency nystagmus. Fundoscopy and red-free fundus imaging were normal. OCT revealed a central foveal thickness of 181 µm in her right eye and 184 µm in her left eye, which is within the normal range for age-based Cirrus measurements.⁴⁴ Foveal morphology was normal, although the outer retinal layers were mildly thinned (Fig. 1A). Goldmann visual field was constricted in both eyes, with a horizontal field of 80° OD and 110° OS for the V4e stimulus. There were also functional central scotomas, represented by a constricted field for the I4e isopter (Supplementary Fig. 1). The LvCCT showed no color discrimination in tritan, deutan, and protan axes, symmetric in both eyes. ERG photopic responses were severely diminished, and scotopic responses were moderately diminished with normal implicit times (Table 1). 

Figure 1.

 

Cirrus OCT and Optos imagining of patients A, C, and D. (A) Macular OCT of patient A, showing normal foveal contour and thickness, yet mildly thinned and “ratty” outer retinal layers. (B) Severe macular atrophy and thinning on patient C. The atrophy is such that we are able to see the choroidal vessels both in color and autofluorescence imaging. (C) Symmetric subfoveal area of lucency affecting the outer layers on patient D. Blunted foveal reflex on color imaging, with a corresponding hypoautofluorescent foveal ring (marked by red arrows OU).

Patient B is an 8-year-old Asian boy who was referred for nystagmus. He described having issues with color vision, glare, and bright lights. His parents did not note changes in his visual function over time. He was 4 years old on his first visit and he has had 4 yearly follow-up visits (Fig. 2A). On his most recent visit (8 years of age), ophthalmic evaluation measured a BCVA of 20/125 OD and 20/160 OS, using red-tinted lenses with mild myopia and astigmatism correction. The patient was orthophoric and presented with low-amplitude, high-frequency nystagmus. Fundus examination showed a bilateral and symmetric mildly blunted foveal reflex. Vessels and periphery were within normality. Central scotomas were noticed on confrontation visual field testing. The LvCCT showed no color discrimination in tritan, deutan, and protan axes, symmetric in both eyes. ERG could not be performed owing to young age and poor cooperation. Although visual acuity, color discrimination and fundus evaluation were stable over the 4 years of follow-up, a subtle subfoveal discontinuity of the outer retinal layers was noticed on his most recent OCT, at age 8 (Fig. 2B). This finding was present in only one cut of the OCT, symmetrically in both eyes. Yet, his central foveal thickness was still normal according to age: 164 µm OD and 162 µm OS.⁴⁴ 

Figure 2.

 

Patient's B longitudinal evaluation. (A) corresponds to his first visit at age 4 in which the macular outer retinal layers were continuous and both color and autofluorescence imaging showed no abnormalities. (B) are images at age 8, in which discontinuity of the outer layers was noted, nasal to the fovea (marked with yellow arrowheads). Both OCT line scans are located within the macula, but alignment is unavailable owing to poor fixation. Color and autofluorescence show none to subtle changes.

Patient C is a 58-year-old Caucasian man who presented for evaluation of cone–rod dystrophy. He reported nonprogressive low visual acuity from birth and absent color vision. His light sensitivity, however, had worsened in the last 10 years. His visual acuity measured 20/200 OD and 20/250 OS. The patient presented with bilateral, symmetric, low-amplitude, high-frequency nystagmus. His retinal examination showed well-circumscribed macular atrophy and nasal peripheral cobblestone atrophic areas bilaterally. His retinal vessels were mildly attenuated and the optic nerve had normal color and cupping. OCT revealed severe macular atrophy with loss of the outer retinal layers (Fig. 1B). Central foveal thickness was decreased at 109 and 102 µm in his right and left eyes, respectively. Goldmann visual field showed constricted field for the isopter I2e and temporal constriction bilaterally (110° horizontal visual field OD and OS for V4e stimulus) (Supplementary Fig. 1). The ERG showed severely decreased amplitude of photopic responses and moderate decrease of scotopic responses, with increased latency (Table 1). 

Patient D is a 52-year-old Ashkenazi Jewish woman who presented with scotomas, color vision issues, and poor visual acuity since she was 16 years old. These symptoms worsened over time, she had undergone two bilateral strabismus surgeries, LASIK, and cataract surgery on both eyes. Her BCVA was 20/200 OD and 20/160 OS, with a myopic correction. She had right exotropia and end-gaze nystagmus in both eyes. Slit-lamp evaluation revealed mild corneal scarring outside the visual axis, possibly related to contact lens use. On fundus examination, the foveal reflex was absent. The retinal vessels were normal and the periphery had myopic pigmentary changes. Autofluorescence imaging revealed a mild hypoautofluorescent foveal ring in both eyes. OCT showed macular thinning with a symmetric hypolucent area affecting the foveal ellipsoid zone (Fig. 1C). Central foveal thickness was decreased: 143 and 136 µm in the right and left eyes, respectively. Goldmann visual field showed constriction of the I3e isopter with mildly constricted field for the V4e stimulus (110° OD and OS, Supplementary Fig. 1). The LvCCT showed no color discrimination in all axes (tritan, deutan, and protan). ERG revealed a flat photopic response and decreased amplitude in scotopic testing (Table 1, Fig. 3). 

Figure 3.

 

Full-field ERG in patient D. Black waveforms correspond to a normal responses. Blue and orange indicate OD and OS in patient D, respectively. Photopic responses are essentially extinguished and the amplitude of scotopic responses is severely decreased as well.

A summary of the ocular findings in our cohort is presented in Table 1. 

Genetic Testing Results

In all four cases, consanguinity was denied, and the family history was negative for ocular disorders. There was no family or personal history of hearing loss, seizures, or any other syndromic features usually associated with inherited eye diseases. 

A summary of the genetic findings in our cohort is described in Table 2. In patient A, we identified two heterozygous putative loss-of-function mutations in PDE6C gene. A previously reported nonsense mutation, NM_006204.4:c.1579C>T; p.Arg527Ter^12,13 was inherited paternally (Fig. 4A). It was located in exon 12, which corresponds with the PDEase (3'5'-cyclic nucleotide phosphodiesterase) catalytic domain (https://www.uniprot.org/uniprot/P51160#family_and_domains accessed on 05/13/2020). This mutation is listed in ClinVar as pathogenic and has an allele frequency of 0.00004377 in gnomAD (https://gnomad.broadinstitute.org/ accessed on 05/13/2020). The second variant is a novel canonical splice donor mutation c.939+2T>G that was inherited maternally, satisfying a compound heterozygous inheritance pattern. It is located downstream of exon 5 and it is affecting the splicing of the GAF2 domain. This variant is absent from ClinVar and HGMD, has an allele frequency of 0.000003985 (gnomAD) and was classified as “Pathogenic” by using the American College of Medical Genetics classification criteria described by Richards et al., 2015.⁴² 

Table 2.

 

Genetic Details of Our Patients’ Variants

 

Figure 4.

 

Probands harboring variants in PDE6C gene. Schematic representation PDE6C gene and protein structure with all of the reported PDE6C variants. (A) Pedigrees of probands, Filled and empty symbols represent affected and unaffected individuals respectively. (B) Illustration of PDE6C gene (NM_006204.4) with novel splicing (this study) and reported splicing/small deletion variants (HGMD). (C) PDE6C protein domain structure (Uniprot_P51160) with identified and reported missense, nonsense and small insertion/deletion variants. GAF (cGMP-specific phosphodiesterases) is a widespread small molecule binding domain, which helps to stabilize catalytic dimer formation. PDEase (3'5'-cyclic nucleotide phosphodiesterase) is important to regulate cAMP catabolism, a vital step in signal transduction pathway. All others represent the following disease phenotypes: autism spectrum disorder, cone dystrophy—early onset, high myopia, Leber congenital amaurosis, major depressive disorder, retinal dystrophy with early onset, and ACHM.

Patients B, C, and D were each homozygous for novel variants of uncertain significance in PDE6C, according to the American College of Medical Genetics classification (Fig. 4, Table 2).⁴² All three variants NM_006204.4:c.2377C>T, p.(Arg793Trp), NM_006204.4:c.1652C>T, p.(Thr551Ile), and NM_006204.4:c.1307C>G, p.(Thr436Ser) are rare in the general population (gnomAD allele frequency 0.00000399, 0.00000397, and 0.00002786, respectively) and predicted to be deleterious by in silico predictions. 

It is necessary for PDE6 proteins to reach their destination in the ciliary compartment of the photoreceptors to perform their function. Variants in PDE6C gene may produce truncated or improperly folded proteins that might restrict its transport toward the rods’ outer segments. An inappropriate amount of functional PDE6 elevates cGMP levels, resulting in rapid retinal degeneration in both humans and animal models.^45,46 The variants in the current study were assessed by conservation analysis and three-dimensional protein modeling (Figs. 5 and 6). The p.Arg793Trp substitution present on patient B is located in the PDEase-I domain and is predicted to diminish intraprotein interactions with other residues (Fig. 5) owing to an increase in hydrophobic forces, which may affect protein folding. However, the missense variant found in patient B [p. (Arg793Trp)] present in catalytic domain, may fold precisely and transport properly to the rods’ outer segments with reduced proteolytic stability, in accordance to similar variants (p.His602Leu and p.Glu790Lys) investigated for functional act by another group.⁴⁶ As per molecular modeling prediction, p.Thr551Ile substitution detected in patient C is proximal to the zinc ion binding site in the PDEase-I domain, which is important for enzymatic activity (Fig. 5). In the same study conducted by Cheguru et al.,⁴⁶ 2015, a nearby variant (p.Met455Val) of PDE6C found in patient A [p. (Arg527*)] and patient C [p.(Thr551Ile)], a nonconserved part of the protein] is reported to transport toward the outer segments, but in a diffused pattern, suggesting a disc rim locality failure. Finally, the missense variant p.Thr436 is part of the GAF B domain, found in cGMP-specific phosphodiesterases and involved in binding of other molecules (Fig. 5). A missense variant (p.Pro391Leu) of PDE6C present in GAF B domain is reported to result in decreased PDE activity in highly significant manner.⁴⁷ In our study, [p.(Thr436Ser)] also resides in GAF B domain and might affect protein activity in a similar way. 

Figure 5.

 

Protein model of PDE6C with structural modeling of PDE6C missense variants. The protein secondary structure is shown in the following colors: helix, yellow; strand, pink; and coil, blue. Residues of interest are shown in forest green color and hydrogen bonding involved residues and bonding pattern are shown in brick red color. Purple color is used to show nearby residues and their distance is drawn with black dashed lines.

Figure 6.

 

Clustal alignment of PDE6C missense variants. Representation of the conservation of amino acids affected by missense variants reported in this study across a number of species.

Discussion

Achromatopsia is typically diagnosed in early childhood in patients with nystagmus, decreased BCVA, and poor color vision.⁴⁸ Parents are often concerned about the visual prognosis and progression of the disease as the child grows. The stationary course of ACHM is an ongoing topic of discussion. This disease is typically characterized as being stable throughout life, although recent papers have shown evidence of progressive cone and even rod involvement in patients with CNGA3,⁵⁰ CNGB3,^49,50 and PDE6C variants.^32,49 In contrast, other groups^51,52 did not find a clear correlation between age and progression of disease. 

We present both children and adults, their cross-sectional data, and the longitudinal evaluation of patient B, followed regularly for 4 years. We note two discernibly different phenotypes in these groups. The children had minor to no structural retinal changes and the adults had macular dystrophy. Although macular dystrophy in adults can be certainly attributed to other common causes such as age-related macular dystrophy or traumatic maculopathy, the presence of a long-standing history of decreased visual acuity as well as the nystagmus on our adult patients led us to think that these structural changes were related with their primary condition, instead of having a superimposed one. Functionally, there were no significant differences among the two groups regarding visual acuity and color discrimination. As Sadowski and Zrenner⁵³ mention, a superimposed rod defect in cone dystrophies can be noticed as a constricted visual field and/or by electrophysiology. The patients who underwent electrophysiologic testing (patients A, C, and D) showed decreased photopic and scotopic responses as well as visual field constriction, allowing us to classify these phenotypes as a cone–rod dystrophy. Interestingly, these patients did not report nyctalopia and preferred mesopic conditions, owing to photophobia. Therefore, cone-derived symptoms may be predominant over the scotopic ones. The progression of the children's phenotype toward the one of the adults remains unknown and could only be answered by a large longitudinal study. The OCT abnormalities noted in patient B during follow-up evaluations support the possibility of progression; however, we are limited in our ability to confirm scan alignment owing to patient cooperation and image quality. However this longitudinal report of a pediatric patient with PDE6C ACHM raises questions about the progressive versus stationary course of this disease. The different phenotypes reported may be part of a continuous spectrum and we believe that as more patients are phenotyped, these questions will be answered. 

All the patients in our cohort presented with novel PDE6C variants, three in homozygosity and one in a compound heterozygous state. In patient A, p.Arg527Ter was previously reported in patients having ACHM, decreased BCVA, and a stable examination, although no information was provided regarding age or retinal findings.^12,13 Its compound heterozygous mutation c.939+2T>G was novel, but a nearby mutation (c.940G>T, p.Glu314Ter) was reported to present with cone–rod dystrophy, although Wang et al.¹⁴ did not disclose further details. 

Our three-dimensional protein analysis of the missense mutation p.Arg793Trp (patient B) predicted a decrease in the normal function of the protein, by decreased interaction with other residues and incorrect protein folding. Functional testing in two other missense arginine to tryptophan variants (Arg29Trp and Arg104Trp) has shown highly significant decrease in PDE activity, by affecting its binding and dimerization.¹¹ One could speculate that the amino acid change from a positively charged arginine to a neutral tryptophan is therefore a significant modification that may, in our case, alter the function of the PDE domain. The patients reported by Grau et al.¹¹ (Arg29Trp and Arg104Trp) had ACHM with essentially normal macula in the fundus evaluation, nystagmus, severe color vision defects and photophobia. Only the age and OCT of the patient carrying the Arg29Trp mutation were reported: 33 years old and a subfoveal optically empty cavity in the ellipsoid zone in both eyes. 

No functional testing has been reported on similar or nearby mutations of patient C's p.Thr551Ile. Adjacent variants (p.Thr546Asn and p.Trp548Leu) only mention ACHM as the phenotype of the patients, with no further information or pictures provided.^13,24 

Mutations near that of patient D's variant (p.Thr436Ser) were classified as pathogenic (e.g., p.Val402Glu, p.Met442Lys, and p.Met455Val).^8,15,22 The phenotype's description of the first two mutations is imprecise, only stating that the patients had ACHM.^15,22 The patient with p.Met455Val variant was 37 years old, had mild pigmentary macular changes, and had a hypolucent area subfoveally by OCT.⁸ 

As mentioned elsewhere in this article and described across the literature, a spectrum of phenotypes has been associated with PDE6C, ranging from ACHM and early-onset cone dystrophy to cone–rod dystrophy and macular atrophy.^{8,30,32,33,49} Most patients with PDE6C variants exhibit ACHM and normal fundus examination or minor macular changes, even as adults.^27,28 Macular atrophy has been reported,^32,33 although this feature is more commonly seen in patients with other affected genes, such as CNGB3.⁵⁴ Our patients’ phenotype correspond with one associated with PDE6C, showing poor color discrimination, decreased BCVA, nystagmus, and macular and cone–rod dystrophy. This finding, along with the specifics on every mutation (in silico tools predictions, gnomAD frequency), makes us call these variants novel disease-causing PDE6C mutations. 

Conclusions

Our cohort provides evidence of rod involvement in patients with PDE6C mutations, which is an atypical presentation for this gene. Additional data and longitudinal points are needed to have accurate information regarding progression and visual prognosis, however our cohort suggests visual acuity and color discrimination may remain stable over time, whereas functional (i.e., rod involvement) changes may present over time. Given that gene therapy for ACHM is currently in the clinical trial phase, knowing when clinical findings appear could narrow the therapeutic window and therefore be a valuable information for both patients and ophthalmologists. Moreover, expanding the spectrum of PDE6C disease-causing mutations improves our understanding of its function and expression as well as the pathophysiology and visual prognosis of this condition. 

Acknowledgments

The authors thank Brett Jeffrey, Delphine Blain, and Amy Turriff for their valuable work with our patients and assistance with this project. 

Supported by the National Institutes of Health – Intramural Research Program. 

Disclosure: M. Daich Varela, None; E. Ullah, None; S. Yousaf, None; B.P. Brooks, None; R.B. Hufnagel, None; L.A. Huryn, None