Novel Bi-allelic PDE6C Variant Leads to Congenital Achromatopsia

Background: The clinical phenotyping of patients with achromatopsia harboring variants in PDE6C has poorly been described in the literature. PDE6C encodes the catalytic subunit of the cone phosphodiesterase, which hydrolyzes the cGMP that proceeds with the hyperpolarization of photoreceptor cell membranes, as the final step of the phototransduction cascade. Methods: In the current study, two patients from a consanguineous family underwent full ophthalmologic examination and molecular investigations including WES. The impact of the variant on the functionality of the protein has been analyzed using in silico molecular modeling. Results: The patients identified with achromatopsia segregated a homozygous missense variant (c.C1775A:p.A592D) in PDE6C gene located on chromosome 10q23. Molecular modeling demonstrated that the variant would cause a protein conformational change and result in reduced phosphodiesterase activity. Conclusion: Our data extended the phenotypic spectrum of retinal disorders caused by PDE6C variants and provided new clinical and genetic information on achromatopsia.


INTRODUCTION
chromatopsia is a congenital visual disorder characterized by the absence of color discrimination, stationary visual impairment, nystagmus, photoaversion, and eccentric fixation [1] . It is a recessively inherited cone dystrophy demonstrable in electroretinogram phototopic responses, with the prevalence rate of 1 in 30,000 [2] . In terms of fundus appearance, normal ophthalmoscopy is mostly expected, although lightly pigmented fundi, minimal granularity of the macula, and less frequently macular defects have been reported [3] .
Potential pathogenic variants in the cone-specific PDE6C gene are associated with achromatopsia. PDE6C (ACHM5, MIM 600827) encodes the catalytic α subunit of cone phosphodiesterase [4] . Upon phototransduction initiation, transducin activates PDE6C through retracting its inhibitory Pγ. Hydrolysis of the cGMP by the active PDE6C results in the closure of the cone outer segment cation channels. It then proceeds with the hyperpolarization of photoreceptor cell membranes, as the final step of the cascade [5] .

Patients
Here, we report a pedigree of our cohort for 63 families with inherited retinal degenerations. After obtaining the precise family history, the patients, a sister and a brother, were examined by an ophthalmologist for confirming the diagnosis of the retinal pathology through extensive ophthalmic workup, including detailed funduscopy, OCT, retinal FA, and electrotretinography.
After gene assessment and variant classification according to the American College of Medical Genetics and Genomics guidance [7], integration of results with patients' phenotype was implemented for providing a clinically relevant interpretation of the findings. Concerning the validity of findings, confirmation of candidate variants and segregation analysis were performed on the two subjects and their parents using the Sanger sequencing method.

Molecular modeling
To understand the possible consequences of the amino acid substitution on the three-dimensional structure of the PDE6C, the residue sequence of the metal-dependent motif of the enzyme catalytic domain was extracted from UniProtKB database through Pfam Database (http://pfam.xfam.org/protein/P51160/). Using Swiss-Model protein homology modeling database (http://swissmodel.expasy.org/interactive/ SgxfJr/models/), the structures of helices H4, H5, H12, H15, H-loop, and M-loop of PDE6C catalytic domain for the wild-type and mutant protein were generated by the application of PDE5/6cd molecule (PDB file: 3JWQ) as the template [8] . Other regions and loops were deleted and incrementally rebuilt into the electron density by manual editing. Chimera tool was used for the visualization and analysis of the modeled protein structure [9] .

Ethical statement
The above-mentioned sampling protocols were approved by the Research Ethics Committee of University of Social Welfare and Rehabilitation, Tehran, Iran (ethical code: IR.USWR.REC1395761). Written informed consents were provided by the patients and their parents.

Clinical evaluation
A brother and a sister aged 28 and 30 at last visit, respectively were descent from first cousin once removed healthy parents (Fig. 1A). The clinical evaluations of the patients are summarized in Table 1. Also, their paraclinic evaluations are indicated in Figure 1C-I in which ophthalmoscopy of the male patient denoted near normal peripheral fundoscopic appearance, although a region of large Lacquer cracks inferior to the macula concordant to pathologic myopia could be observed. Despite similar macular changes in the fundoscopy of the female patient, it disclosed much more findings, including large optic disc, tilted optic nerve with peripapillary atrophy, peripapillary crescent and drusen, tigroid-blonde fundus, Lacquer cracks, and Dalen-Fuchs nodules, all resembling bilateral pathologic myopia. The 30-hz flicker response in electroretinogram revealed non-recordable cone function with subnormal rod function. Furthermore, SD-OCT indicated that the index patients were categorized into stage V, according to the SD-OCT features and staging system categorized by Greenberg et al. [10] , defined as complete RPE disruption and loss of the ONL with choroidal hyperreflectance. Moreover, the FA images demonstrated a transmission, or window defect, due to missing RPE that normally blocks fluorescence from choroid. As a result, the bright choroidal fluorescence evidenced the complete RPE disruption. The parents reported no visual disturbance, and also they demonstrated to be normal when undergoing a comprehensive ophthalmic examination.

PDE6C screening
Using bioinformatics filtering strategies, a novel homozygous missense variant in PDE6C (NM_006204:chr10:g.95400714C>A:c.1775C>A:p.A5 92D) was identified in the proband. Co-segregation analysis confirmed the result (Fig. 1B). The variant, p.A592D, initially was classified as a variant of uncertain significance based on the American College of Medical Genetics and Genomics guidelines. However, there were some indicators of the pathogenicity of the variant as follows: (a) located in a critical functional domain (metal-dependent motif of the enzyme active site), (b) representing ultra-rare allele being absent in general population alleles, (c) predicted to be deleterious and evolutionary conserved, and (d) co-segregated with the achromatopsia phenotype in the family. Apart from heterozygous parents, two cousins were also analyzed as the patients without unaffected sibs. On top of that, considering the complete analysis of 269 genes registered in the RetNet database, specifically focusing on cone-rod dystrophy relevant genes and no opposing arguments regarding pathogenicity, its causality appeared to be pertinent.

Protein structure analysis
The PDE6C metal binding motif sequence showed 53.3% sequence identity to PDE5/6cd as a structural template to model residues 562-606 of PDE6C (Fig.  2). The p.A592D variant converts alanine (A) with hydrophobic and aliphatic moiety to aspartic acid (D) with acidic side chain that differs in size and electric charge.

DISCUSSION
This report describes two affected siblings with achromatopsia and a novel likely pathogenic PDE6C variant in the family. The product of this gene consists of a couple of cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA domains, followed by a catalytic domain. The p.A592D variant was located within the highly conserved HD motif with a predicted metal cation (Zn 2+ and Mg 2+ )-dependent phosphorhydrolase activity at the catalytic domain with a pivotal role in signal transduction, according to SMART protein domain annotation resource [11] . The A592 residue was fully conserved among vertebrates. Thus, it is assumed that the substitution introduces a negative charge in buried alanine, which probably would not fit in the core of the protein and might lead to conformation collision and folding defects [12] . Intriguingly, the conformation change could abrogate Zn 2+ binding, thus potentially preventing the enzyme catalytic activity. Functional analysis of molecular modeling regarding an amino acid alteration just before A592 (E591K) illustrated that it likely decreases metal cation attachment [13] . On the other hand, the outcome of this modeling analysis was concordant with the results of a structural study of phosphodiesterase inhibition by the C-terminal region of the γ subunit. It demonstrated that Pγ docking site is juxta-positioned to H-loop, H12, M-loop, and H15, residues 609-632, 674-690, 747-771, and 772-793, respectively [8] . Our molecular modeling showed that the conformational change in H5 would interfere with Pγ functionality (Fig. 2G-H). It has also been indicated that obliteration of Pγ in mouse photoreceptors significantly reduces PDE6C hydrolyses and leads to retinal degeneration, despite the expectation that the removal of the inhibitory Pγ subunit from holoPDE6C would activate the enzyme [14] . It has been hypothesized that once the catalytic pockets were concealed by the Pγ subunits, aryl hydrocarbon receptor-interacting protein Like 1, as an obligate chaperone of PDE6C, would change the folded structure into "open" conformation [8,12] . Therefore, it could be concluded that the A591D variant might interfere with the activity of PDE6C phosphodiesterase.
Loss of function mutations of PDE6C decreases intracellular cGMP hydrolysis. Increased level of cGMP primarily causes the excessive opening of cGMP-gated channels in cone outer segment, resulting in an unrestricted influx of Ca 2+ . It has been demonstrated that uncontrolled cytoplasmic elevation of cGMP and Ca 2+ cations contributes to the demise of cones [15] . Therefore, loss of function of PDE6C will give rise to achromatopsia.
There are quite limited published data on PDE6Crelated retinal phenotype [6,16,17] . The HGMD has currently listed 38 variants in PDE6C that explain the disease phenotype in patients. Our cases presented with the typical features of achromatopsia since birth. Apart from pathologic myopia, fundus examination revealed atrophic chorioretinal macular corresponded to an area of complete loss of autofluorescence on FA imaging. FA signal with window defect highlights the complete RPE disruption, in keeping with stages five of the OCT classification proposed by Greenberg et al. [10] recently that shows severe macular thinning of all layers and excavation in retina and choroid of both eyes. Electroretinogram also confirmed complete degeneration of cones. However, the scotopic response was also concordant with subnormal rod function. Although PDE6C is exclusively expressed in cones, this photoreceptor-specific defect would lead to a series of cellular changes, ultimately affecting the normal function of the whole retina [18] .
The novel PDE6C variant (p.A592D) in a homozygous state could explain a part of the phenotypes in our cases with achromatopsia. However, it is insufficient to explain the fundoscopic differences between the two patients. It could be explained that there might be other potentially relevant variants involved in the female index. As the WES technology was only performed in the male proband, detection of other pathogenic variants causing severe myopia was inapplicable.
In this study, we identified a novel PDE6C variant in two cases of achromatopsia with macular atrophy. Our data extended the phenotypic spectrum of retinal disorders caused by PDE6C variants and provided new clinical and genetic information.