Increased Plasma Cgmp in a Family with Autosomal Recessive Retinitis Pigmentosa Due to Homozygous Mutations in the PDE6A Gene

Retina Increased Plasma cGMP in a Family With Autosomal Recessive Retinitis Pigmentosa Due to Homozygous Mutations in the PDE6A Gene

Ulrika Kjellstr¨om,1 Patricia Veiga-Crespo,2 Sten Andreasson,´ 1 and Per Ekstr¨om2

1Lund University, Skane University Hospital, Department of Clinical Sciences Lund, Ophthalmology, Lund, Sweden 2Lund University, Faculty of Medicine, Department of Clinical Sciences Lund, Ophthalmology, Lund, Sweden

Correspondence: Ulrika Kjellström, PURPOSE. To describe genotype and phenotype in a family with autosomal recessive retinitis Ogonkliniken¨ Sk˚anes Universitetss- pigmentosa (arRP) carrying homozygous mutations in the gene for the a-subunit of cyclic jukhus Lund, S 221 85 Lund, Swe- guanosine monophosphate (cGMP)–hydrolyzing phosphodiesterase 6 (PDE6A). Moreover, to den; compare their plasma cGMP levels to controls, exploring the possible role for cGMP in RP [email protected]. diagnostics. Submitted: May 4, 2016 Accepted: October 10, 2016 METHODS. Seven siblings and their parents were recruited. Microarray, verified by Sanger sequencing, was used for genotyping. Investigations included slit lamp and fundus Citation: Kjellström U, Veiga-Crespo P, examination, Goldmann perimetry, full-field and multifocal electroretinography (ERG), and Andreasson´ S, Ekström P. Increased optical coherence tomography (OCT). Cyclic GMP was measured with an immunoassay kit. plasma cGMP in a family with autosomal recessive retinitis pigmentosa due RESULTS. All siblings and their father were homozygous, and the mother heterozygous, for to homozygous mutations in the IVS6þ1G>AinPDE6A. Seven family members also carried c1532G>AinABCA4. Visual fields PDE6A gene. Invest Ophthalmol Vis were constricted with mere central remnants in older subjects and additional temporal Sci. 2016;57:6048–6057. DOI: crescents in younger subjects. Visual acuity ranged from 0.8 to amaurosis. Full-field ERGs 10.1167/iovs.16-19861 showed extinguished rod responses and minimal cone responses. Multifocal ERGs were severely reduced. Optical coherence tomography revealed either general attenuation or central macular edema. Mean plasma cGMP in patients was approximately twice that in controls.

CONCLUSIONS. To our knowledge, this is the ﬁrst phenotypic description of arRP due to homozygous IVS6þ1G>A mutations in PDE6A and these seem here to be associated with severe RP leading to early extinction of rod responses as well as reduced macular function. Additionally, patients showed increased plasma levels of cGMP, indicating a possible role for cGMP measurements as part of the clinical tests for this and, after further investigations, maybe other forms of RP. Keywords: cyclic GMP, electroretinography, retinitis pigmentosa

etinitis pigmentosa (RP) belongs to the group of hereditary although a strategy for cases with RP mutations concerning the R retinal degenerations. The prevalence of RP is approximate- synthesis of certain lipids has been put forward.9 ly 1 in 4000 persons worldwide1 and it is a major cause of Despite the diverse genetic origin of RP, fundus findings progressive deterioration of vision and blindness among most often share a common pattern with pale optic nerve head, younger people.2 The disease typically starts with loss of night attenuated retinal blood vessels, and bone-spicule pigmenta- 1 vision in adolescence, followed by visual field constriction in tions in the midperiphery or toward the far periphery. Those young adulthood and gradual reduction of central visual acuity morphologic changes are preceded and accompanied by later in life.1 The most common mode of inheritance is photoreceptor cell death starting with rod photoreceptors autosomal recessive (ar) (50%–60% of cases), but RP can also and secondarily also engaging cones, which are considered to get damaged and die from oxidative stress,10–12 loss of be inherited in an autosomal dominant (30%–40%) or X-linked 13 3,4 metabolic and trophic support, or toxicity due to rod cell (5%–15%) way. At the moment, more than 60 genes associated 14 5 death. Indeed, alterations in antioxidative status have been with RP have been identified (see also: https://sph.uth.edu/ demonstrated in peripheral blood of RP patients,15 with retnet/home.htm; provided in the public domain by The reduced activity of superoxide dismutase 3 and increased University of Texas Health Science Center, Houston, Texas, levels of nitric oxide (NO). This imbalance in antioxidant status USA). Many of these genes code for proteins that are involved in may contribute to a poorer capacity of RP patients to cope with the phototransduction cascade, the retinoid metabolism, or in toxic oxygen metabolites.15 the maintenance of photoreceptor integrity.6 To date, RP cannot The small second messenger molecule cyclic guanosine be cured, although gene therapy has been tested for some monophosphate (cGMP) plays an important role in the retinal genetic variants.7,8 There are also no established biochemical phototransduction cascade, which is initiated by a conforma- blood markers yet to contribute to the clinical test possibilities, tional change of the photoreceptor opsins after getting hit by

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the photons of light. Subsequently, transducin levels are elevated, thereby activating the rod photoreceptor–specific cGMP–phosphodiesterase-6 (PDE6), which, in turn, catalyzes the breakdown of cGMP. Decreased cGMP levels lead to closure of cyclic-nucleotide–gated (CNG) cation channels and reduction of Ca2þ and Naþ influx,16 and the following hyperpolarization results in signal transmission to second- order neurons.17 Apart from the physiologic importance in phototransduction, PDE and cGMP are likely also involved in pathologic events, since high cGMP levels, and subsequent photoreceptor cell death, are encountered in several animal RP models based on mutations of PDE6, and interestingly also as a consequence of mutations in apparently unrelated genes.18–21 The PDE6 enzyme is composed of two catalytic subunits called a (A) and b (B) and two identical inhibitory c subunits.22 Approximately FIGURE 1. Pedigree for the family. All family members except for I-2 3% to 4% of arRP cases21,23 are caused by mutations in the are homozygous for IVS6þ1G>A (c.998þ1G>A), a splice site mutation, PDE6A gene, coding for the a subunit and resulting in a in the PDE6A gene. defective PDE6 enzyme or no enzyme at all. Mutations in the PDE6B gene, coding for the b subunit of PDE6, are likewise further exploration of the possible connections between RP frequent and also lead to retinal degenerations.24–26 The PDE6 and the cGMP system in compartments outside of the retina, mutations, as well as others that lead to increased cGMP levels, not the least at the clinical level, since the possibility to use may cause an undesired opening of CNG cation channels, blood cGMP as a diagnostic tool for the disease would be most allowing continuous and pathologic Ca2þ influx with subse- welcome. To this end we here studied retinal function and quent rod photoreceptor cell death.27,28 Another possibility is morphology in a consanguineous family with arRP and that the increased cGMP levels cause cell death via activation identified their genetic errors as homozygote mutations in of cGMP-dependent protein kinase.29 Of the PDE6A and the PDA6A gene and, in some cases, also heterozygote or PDE6B mutations, the most extensive clinical descriptions so homozygote mutations in ABCA4.Thispermittedusto far come from PDE6B families, while descriptions of the describe several aspects of the ophthalmologic status of phenotype associated with PDE6A mutations are less frequent. patients with homozygote PDE6A mutations, which has not Another gene that can be associated with arRP is the ABCA4 been done to such an extent previously. In addition, it gave us gene,30–32 most often encountered in Stargardt disease33–35 but the opportunity to measure blood cGMP levels and to compare also seen in other retinal degenerations. The ABCA4 gene them to those of healthy controls, in a situation where we codes for the ABCA4 protein, an ATP-binding cassette (ABC) know that the cGMP system is involved in the disease. transporter protein located in the rim of the photoreceptor discs.36–38 ABCA4 promotes the clearance of toxic vitamin A metabolites such as N-retinylidene-phosphatidylethanolamine METHODS and phosphatidylethanolamine from the lumen of the outer segment disc membranes during phototransduction.39,40 De- Subjects and Controls fective function or absence of ABCA4 results in a buildup of the lipofuscin fluorophore N-retinylidene-N-retinylethanolamine, In this study, a large consanguineous family of Iraqi origin was which then accumulates in RPE cells, leading to RPE cell investigated. Seven siblings with nonsyndromic arRP and their death and probably secondary loss of photoreceptors.40,41 It parents were included (pedigree, Fig. 1). Four of the subjects has also been proposed that direct photoreceptor cell death (II-4–II-7) had also been examined at our Department of may precede RPE alterations.42,43 Ophthalmology, Sk˚ane University Hospital, Lund, Sweden, at a Given the fact that cGMP is an important retinal player in previous occasion in 2004. One of them, II-4, could not be general, and also in RP at least when PDE6A and PDE6B reexamined for the current study. The mean age of family mutations are concerned, clinical measurements of cGMP members was 43 6 16 years (range, 28–73 years; Table). The levels in ocular tissue or in body fluids in various retinal states study was conducted in accordance with the tenets of the are few. In the context of retinal detachment, La Heij et al.44 Declaration of Helsinki and it was approved by the Ethical have shown reduced cGMP levels in vitreous as well as Committee for Medical Research at Lund University. All subretinal fluid in patients with detached retinas, compared to subjects gave their informed consent to participate. control samples, and similar results have been obtained in Plasma cGMP concentration was also measured in 20 experimental retinal detachments in pigs.45 The reduced cGMP nonsmoking healthy volunteers without any known eye levels may have been due to lowered production of cGMP in disease or renal problems. Controls were aged 41 6 6 (range, the affected retinal cells.44,45 Interestingly, with respect to RP, a 30–50) years. study by Martinez-Fernandez de la Camara and colleagues15 reports elevated cGMP blood levels in RP patients. However, Genetic Analysis the authors do not discuss this in relation with high photoreceptor cGMP, but rather use the blood cGMP as an DNA was extracted from venous blood drawn from the index of the activity of the NO/cGMP pathway and of oxidative precubital vein in all subjects. The DNA was screened on an stress as such. Moreover, the report does not describe the RP arRP genotyping microarray test for 710 mutations in 28 patients with respect to mutation. known arRP genes (CERKL, CNGA1, CNGB1, MERTK, PDE6A, We thus have a situation where experimental studies PDE6B, PNR, RDH12, RGR, RLBP1, SAG, TULP1, CRB, RPE65, suggest a mechanistic cGMP–RP connection, and where USH2A, USH3A, LRAT, PROML1, PBP3, EYS, ABCA4, AIPL1, Martinez-Fernandez de la Camara et al.15 have found higher CNGA3, CNGB3, GRK1, IMPG2, RHO, and RP1). Testing was blood cGMP in RP patients; moreover, we know that some performed at Asper Biotech (Asper Ophthalmics, Tartu, patient cohorts indeed carry mutations in PDE6. This warrants Estonia) (http://www.asperbio.com/asper-ophthalmics/

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OCT, Rod ffERG Cone ffERG Subject/Age, ABCA4 BVA, RE, LE, Goldmann Macular Ampl, Ampl, mERG Ampl, cGMP Conc, y Sex Mutation 2004 BVA, RE, LE Visual Field Map lV, RE, LE lV, RE, LE lV nM

I-1/73 Male c.1532G>A het Not tested P, amaurosis Not tested Not tested Extinguished Residual Not tested 150 responses 0.5, 1 I-2/68 Female c.1532G>A het Not tested Not tested Not tested Not tested Not tested Not tested Not tested Not tested II-1/46 Female c.1532G>A hom Not tested 0.1, 0.16 V4e < 108, I4e < 58 Central macular edema Extinguished Residual 1 170 with vitreoretinal responses traction, otherwise 0.5, 2 attenuation II-2/45 Male No mutation Not tested 0.2, 0.2 V4e 128–138, II4e Central macular edema Not tested Not tested Not tested Not tested 28–48 with vitreoretinal traction, otherwise attenuation II-3/42 Female c.1532G>A hom Not tested 0.32, 0.5 V4e < 108, I4e < 58 General attenuation Extinguished Residual 1 171 responses 4, 1.5 II-4/36 Female c.1532G>A het 0.4, 0.6 Not tested RE V4e 158,LE208 Not tested Extinguished Residual Not tested Not tested and a temp responses crescent from 1, 1 508–808, I4e 58 II-5/34 Female c.1532G>A het 0.8, 0.4 0.3, 0.25 RE V4e from 158 nas Central macular edema, Extinguished Residual 1 154 to 908 temp but otherwise attenuation responses with a scotoma, 0.5, 1 I4e 58, LE V4e 108 and a temp crescent from 608–858, I4e 58 II-6/34 Female No mutation 0.3, 0.04 0.13, 0.1 V4e < 108 and a General attenuation Extinguished Residual 1 202 temp crescent responses IOVS 708–908, I4e < 58 1, 1 j

II-7/28 Female c.1532G>A het 0.9, 1.0 0.25, 0.8 V4e l58–208 and a RE central macular Extinguished Residual 1 146 2016 November temp crescent edema otherwise responses 508–908, I4e 78– attenuation, LE 1, 0.5 108 general attenuation Normal 277 6 72 63 6 20 28 6 6836 26 values

47 j

For comparison we also give ERG data that can be considered to reﬂect the normal population. The control values are based on parts of previously published material as well as supplemented by 57 Vol. ERG measurements in some additional normal subjects to adjust the control material to the age span of the present patient group (for ffERG, 34 control subjects aged 22–67 years [mean 42 years]; and for mERG, 25 control subjects aged 25–69 years [mean 43 years]). Ampl, amplitude; BVA, best visual Snellen acuity; Conc, concentration; het, heterozygote; hom, homozygote; LE, left eye; nas, nasal; P,

light perception; RE, right eye; temp, temporal; , reduced. j o 14 No. j 6050 cGMP Plasma Levels in RP Due to PDE6A Mutations IOVS j November 2016 j Vol. 57 j No. 14 j 6051

autosomal-recessive-retinitis-pigmentosa-genetic-testing/ tations in the periphery. In addition, pigmentary changes were autosomal-recessive-retinitis-pigmentosa-apex-based-test- found in the macular regions, and II-6 had a macular scar in her details; provided in the public domain). Microarray results left eye. The extent of peripheral pigmentations increased were verified by Sanger sequencing. with age as expected. Figure 2 shows fundus photos (Figs. 2a, 2e) from II-3 and II-7, representing one elder and one younger Ophthalmologic Examination sibling. All subjects except for the youngest, II-7, had some degree of nuclear cataract. II-1 and II-3 had both had cataract Slit lamp and fundus examinations were performed in all surgery and internal limiting membrane peeling in their right subjects. Best corrected monocular visual acuity (BCVA) was eyes in 2010, followed by YAG laser capsulotomy in 2012. tested on the Snellen chart at 5 m. Visual fields were mapped These surgical procedures were performed in clinics abroad. by using a Goldmann perimeter with standardized objects V4e II-5, II-6, and II-7 all had slight nuclear lens opacities. and I4e. In one subject (II-5; see pedigree, Fig. 1), the III4e Best corrected monocular visual acuity ranged from object had to be used instead of I4e at the first visit, since she amaurosis in the eldest family member (I-1) to 0.8 Snellen in was not able to identify a smaller object. the youngest (II-7) (Table). In the subjects that had been examined in 2004 (II-4–II-7), a deterioration of BVCA was Full-Field Electroretinography found (Table). Visual fields were constricted to approximately 158 to 108 or In 2004, full-field electroretinograms (ffERGs) were recorded in some cases even less, for the V4e object in all siblings with a Nicolet Viking analysis system (Nicolet Biomedical (Table). The younger ones (II-4–II-7) also had temporal 46 Instruments, Madison, WI, USA) as described previously and crescent-shaped remnants of varying extent left (see also Fig. 2 in 2014, ffERGs were recorded with an Espion E analysis 3 for representative visual fields from siblings II-3 and II-7; system (Diagnosys, Lowell, MA, USA). Table). In two of the subjects, who were also examined in 2004 (II-5 and II-7), the visual fields showed approximately 108 of Multifocal Electroretinography further constriction for the V4e object on the last examination compared to the first (V4e approximately 208–258). In subject Multifocal ERGs (mfERGs) were registered with a Visual II-6, the central parts of the visual fields were stable between Evoked Response Imaging System (VERIS; EDI, San Mateo, 46 the two investigations, but on the last one, a temporal crescent CA, USA) according to previous descriptions. not previously shown was identified.

Optical Coherence Tomography Full-Field Electroretinography Macular thickness was measured with a Topcon 3D OCT-1000 47 Full-field ERGs were recorded in six of the siblings (II-1, II-3–II- (Topcon, Inc., Paramus, NJ, USA) as previously described. 7). All of them showed extinguished rod responses, while they still had minimal residual cone responses measuring approx- Cyclic GMP Measurement imately 1 lV in amplitude (Table). The same results were found for II-4 to II-7 when they were examined in 2004. All blood sampling was performed at same time of day (noon). The blood was collected in identity-coded heparin tubes and kept on ice until centrifugation at 1750g in a SkySpin CM-6MT Multifocal Electroretinography centrifuge (ELMI Ltd, Riga, Latvia), to enable the plasma fraction Multifocal ERGs were obtained in five of the siblings (II-1, II-3, to be collected. The samples were then kept at 808 C until the II-5–II-7) and revealed severely reduced cone responses in all cGMP measurement. Possible circulating, systemic PDEs were rings of the mERG (Table). Data from II-3 and II-7 are shown in inactivated after the addition of 1/10 volumes of 1 M HCl. If Figure 2. some precipitation occurred, the acidified plasma was centri- fuged again, and the supernatant transferred to a clean tube. The measurement of the cGMP was carried out by using the Optical Coherence Tomography cGMP Direct Immunoassay kit (Abcam ab65356; Abcam, Optical coherence tomography scans were captured in II-1 to Cambridge, MA, USA), following the manufacturer’s instruc- II-3 and II-5 to II-7. The OCT scans showed either severe tions for the kit, with samples being acetylated before general attenuation in the posterior pole (II-3, II-6 and II-7 left measuring. The measurements were carried out at k450nm with eye) or central macular edema accompanied with peripheral an ELISA plate reader (SPECTROStarNano; BMG Labtech, attenuation (II-1 and II-2, II-5, II-7 right eye). Examples of OCT Ortenberg, Germany). The absolute values of the samples scans for II-3 and II-7 are shown in Figure 2. were calculated by comparing measured values to a standard curve made up from pure cGMP, as provided in the kit. Genotype Statistics All siblings (II-1–II-7) and their father (I-1) were homozygous for IVS6þ1G>A (c.998þ1G>A), a splice site mutation, in the Statistical analyses were performed in SPSS 20.0 (IBM Corp., PDE6A gene. The mother (I-2) was heterozygous for the same Armonk, NY, USA). Statistical significance was defined as P < mutation (Fig. 1). Moreover, subjects II-1 and II-3 were 0.05. Owing to the small samples sizes, a nonparametric homozygous for the c.1532G>A variant in the ABCA4 gene statistical test (Mann-Whitney U test) was used. and subjects I-1, I-2, II-4, II-5, and II-7 (Table) were heterozygous for the same mutation, which is a missense mutation leading to a substitution of His for Arg at position RESULTS 511. It is quite uncommon but it has once, in the compound Ophthalmologic Examination heterozygote state, been described to lead to a Stargardt phenotype.48 Although ABCA4 mutations are known to cause All siblings had typical RP fundus findings with pale optic arRP,30–32,49–51 in this setting the PDE6A mutation seemed to discs, narrowed retinal vessels, and bone corpuscle pigmen- be the causative mutation considering the fact that all family

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FIGURE 2. Fundus photos, OCT images, and mfERGs from two representative siblings, one of the older, II-3, and one of the younger, II-7. In both (a, e) the fundus photos show pale optic discs, attenuated retinal vessels, and peripheral bone corpuscle pigmentations, which are more widespread in the older sibling. In II-3 the OCT images show attenuation (b, c) and the mfERGs are severely reduced (d) as compared to normal (i). In II-7 the OCT images reveal central retinal thickening (f) due to macular edema (g), and macular function measured with mfERGs is severely reduced (h)as compared to normal (i).

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FIGURE 3. Representative Goldmann visual fields from one elder sibling, II-3 (top), and one younger, II-7 (bottom). II-3 has small residual visual fields with V4:e constricted to less than 108 and I4:e to less than 58. II-7 has somewhat larger central remainders of the visual fields as well as spared temporal crescents for V4:e.

members with the homozygous IVS6þ1G>A (c.998þ1G>A) In the literature, normal plasma/serum cGMP levels, PDE6A mutations had the same severe retinal dysfunction, no obtained by various methods, have often been reported in matter if they were homozygous, heterozygous, or lacked the the lower nanomolar range, although values up to several ABCA4 mutation (c.1532G>A) (Table). Nor did there seem to hundred nanomolar have also been given.56–63 Our control be any association with the presence of ABCA4 mutations and cGMP plasma levels at approximately 80 nM therefore macular morphology or function, although ABCA4 mutations compare relatively well with this. most often are related to Stargardt disease.33–36,52–55 Macular edema was encountered in a subject without the c.1532G>A ISCUSSION ABCA4 mutation (II-2) as well as in family members who were D heterozygous (II-5 and II-7) or homozygous (II-1) for the We have studied a consanguineous family with a severe form of c.1532G>A mutation. The same lack of association to the arRP and report several ophthalmologic data consistent with ABCA4 mutations was shown for macular attenuation with no extinguished rod responses and markedly reduced cone ABCA4 mutation in subject II-6, a heterozygous c.1532G>A function by the early twenties. We also present information mutation in subject II-7, and homozygous c.1532G>A muta- on a possible disease-related deviation from normal plasma tions in subject II-3. cGMP levels in these patients. To the best of our knowledge, this is the first phenotypic Cyclic GMP Measurement description of arRP due to homozygous IVS6þ1G>A (c.998þ1G>A) mutations in the PDE6A gene and includes Plasma cGMP concentration was measured in six of the family the somewhat unique situation where all family members members (I-1, II-1, II-3, II-5–II-7). The mean cGMP level was (except the mother) show such homozygosity as well as a 166 6 19 nM (mean 6 SD) and the range, 146 to 202 nM. rather uniform phenotype. The IVS6þ1G>A (c.998þ1G>A) Cyclic GMP in the eldest subject, the father I-1, was 150 nM mutation has been confirmed as a novel splice site mutation in (Fig. 4a; Table). patients with arRP23 and it is considered to be a null allele, a Mean plasma cGMP in control subjects was 83 6 26 (range, fact that may explain the severe phenotype in our subjects, 40–157) nM. When analyzed for statistical significance, the although PDE6A-associated arRP sometimes has been de- values from the PDE6A family members were found to be scribed as a milder form of arRP.24,26 All of the subjects, significantly higher than those of the control group (P ¼ 0.001). regardless of age at examination, also had severely reduced A receiver operating characteristic (ROC) curve was also macular function as measured by mfERG, and OCT analyses established, which revealed that the cGMP test has a good showed either macular attenuation or edema with, or without, sensitivity, but a somewhat lower specificity (Fig. 4b). vitreoretinal traction. Macular edema and vitreoretinal traction

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FIGURE 4. (a) Plasma cGMP concentration for controls (n ¼ 20) and RP patients (disease) (n ¼ 6). Boxes show median and interquartile range, while bars illustrate range and circles indicate outliers. (b) The ROC curve (i.e., the blue curve in the figure) shows the sensitivity in relation to the reciprocal of the specificity (i.e., 1-the specificity, sometimes denoted as the false-positive rate). The green diagonal line is a reference line representing a situation where no prediction at all can be made from the presumptive indicator. This ROC curve indicates that the cGMP test has good sensitivity, that is, it can find diseased patients very well, but with a bit lower specificity, that is, carrying a risk that some nondiseased subjects may appear as having the disease (false positive).

have been described in arRP patients with other mutations in dependent on melatonin levels.62 With a possible connection the PDE6A gene24,26 and may contribute to the early reduction to this, the ROC specificity aspect indicates that some healthy of central visual function. For example, the youngest sibling in subjects may appear as having the disease. Yet, we find it our study had reduced visual acuity (0.25) in her right eye with compelling that subjects with a mutation in PDE6A, a gene macular edema, compared to her left eye showing macular coding for a cGMP-hydrolyzing enzyme highly expressed in the attenuation but still quite good visual acuity (0.8). The retinal photoreceptors,65 present with high levels of the phenotype was also characterized by presenile nuclear cataract nonhydrolyzed substrate as well as with reduced photorecep- in all but one sibling (II-7, the youngest). tor function. We are currently analyzing whether retinas from An aspect that would potentially complicate the interpre- animal RP models do release their high cGMP into culture tation of genotype–phenotype correlation in this family was medium, and note that this seems to be the case (unpublished the additional presence of ABCA4 mutations (c.1532G>A), results, but see Lolley et al.66). It is hence possible that the lack either heterozygously (I-1, I-2, II-4, II-5, and II-7) or homozy- of cGMP hydrolysis results in increased extracellular cGMP that gously (II-1 and II-3), in some family members (Table). In this manages to find its way to the circulation. One may argue that setting, though, the PDE6A mutation seems to be causative, patients with a loss of measurable retinal functions as in this since all family members with the homozygous IVS6þ1G>A case should not have any photoreceptors left to produce cGMP. (c.998þ1G>A) PDE6A mutations had the same severe retinal However, results from a PDE6A mutant canine model suggests dysfunction, whether or not they carry the ABCA4 mutation that ERGs may be severely affected well in advance to (c.1532G>A) (Table). A similar situation with a well-known structural loss of photoreceptors,67 and the present clinical rhodopsin mutation in all family members and an extra finding does not as such exclude the presence of rod mutation in the gene for cGMP-gated channels in some siblings photoreceptors, albeit in reduced numbers. Moreover, our has been described by Dryja et al.,64 who concluded that the results agree with those of Martinez-Fernandez de la Camara et shared rhodopsin mutation had to be the cause of retinal al.15 where the serum cGMP levels of a group of RP patients are degeneration, while the other mutation was carried only by increased by approximately 65% compared with a control chance and without apparent effects. group.15 In the latter study, the RP mutations in question are The difference in plasma cGMP levels between RP patients not reported, however, and at least some may thus be and controls (RP twice that in controls) is very clear, and the unrelated to PDE6 gene mutations as such, which would corresponding ROC curve suggests that the cGMP test readily appear inconsistent with our conclusions. On the other hand, finds diseased patients. Still, at this point we cannot claim that as mentioned in the Introduction, several RP models with the higher level is a direct consequence of the disease. mutations in genes, including but not only PDE6, display high Differential blood cGMP levels have previously been seen in a photoreceptor cGMP, suggesting this is a common denomina- variety of situations, for instance, in connection with tor for a number of RP types,18,21,68 although at this point we natriuretic drugs for acute heart failure,58 blood pressure– do not know the underlying mechanisms. Since one may altering drugs,59 preeclampsia,60 migraine,61 and in relation to expect high similarity between photoreceptor pathology of the circadian rhythm,63 where the cGMP levels may in turn be human homologue models and that of RP patients, high cGMP

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in photoreceptors, and possibly in the circulation, might 7. Jacobson SG, Cideciyan AV, Aguirre GD, et al. Improvement in therefore appear also in larger patient groups, independently of vision: a new goal for treatment of hereditary retinal the exact mutation. degenerations. Expert Opin Orphan Drugs. 2015;3:563–575. It appears unlikely that a plasma cGMP test on its own 8. Jacobson SG, Cideciyan AV, Ratnakaram R, et al. Gene therapy would be used to screen parts of the general population with for leber congenital amaurosis caused by RPE65 mutations: respect to detecting RP, not the least because there could be a safety and efficacy in 15 children and adults followed up to 3 considerable risk for false positives. The latter may occur years. Arch Ophthalmol. 2012;130:9–24. because of a cGMP connection to other clinical or nonclinical 9. Wen R, Lam BL, Guan Z. Aberrant dolichol chain lengths as situations, as mentioned above, and in this context there are biomarkers for retinitis pigmentosa caused by impaired reports suggesting PDE6 expression also in nonphotoreceptor dolichol biosynthesis. J Lipid Res. 2013;54:3516–3522. tissues and in different developmental stages,69 at least in other 10. Catala A. Lipid peroxidation of membrane phospholipids in animals, which may have a bearing on the plasma cGMP levels. the vertebrate retina. Front Biosci (Schol Ed). 2011;3:52–60. Still, the measurement of plasma cGMP is relatively simple and 11. Komeima K, Rogers BS, Lu L, Campochiaro PA. Antioxidants could readily act as a complement to other ophthalmic reduce cone cell death in a model of retinitis pigmentosa. Proc investigations in order to improve the RP diagnosis, which is Natl Acad Sci U S A. 2006;103:11300–11305. not always straightforward. Such cGMP analyses could be 12. Shen J, Yang X, Dong A, et al. Oxidative damage is a potential envisaged to take place in connection with regular eye cause of cone cell death in retinitis pigmentosa. J Cell Physiol. examinations of defined or suspected RP patients, including 2005;203:457–464. family members. There are of course a number of questions 13. Leveillard T, Mohand-Said S, Lorentz O, et al. Identification and remaining before this can be reached, the most important characterization of rod-derived cone viability factor. Nat being whether the blood cGMP–RP connection is real. We Genet. 2004;36:755–759. therefore plan repeated analysis of cGMP in more RP patients 14. Punzo C, Xiong W, Cepko CL. Loss of daylight vision in retinal with other causative mutations. degeneration: are oxidative stress and metabolic dysregulation In conclusion, this is, to our knowledge, the first study that to blame? J Biol Chem. 2012;287:1642–1648. describes the phenotype in arRP due to the IVS6þ1G>A 15. 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