Novel Variants in Phosphodiesterase 6A and Phosphodiesterase 6B Genes and Its Phenotypes in Patients With Retinitis Pigmentosa in Chinese Families

Background: Retinitis pigmentosa (RP) is a genetically heterogeneous disease with 65 causative genes identied to date. However, only approximately 60% of RP cases genetically solved to date, predicating that many novel disease-causing variants are yet to be identied. The purpose of this study is to identify novel variants in phosphodiesterase 6A and phosphodiesterase 6B genes and present its phenotypes in patients with retinitis pigmentosa in Chinese families. Methods: Five retinitis pigmentosa patients with PDE6A variants and three with PDE6B variants were identied through a hereditary eye disease enrichment panel (HEDEP), all patients’ medical and ophthalmic histories were collected, and ophthalmological examinations were performed, then we analysed the possible causative variants. Sanger sequencing was used to verify the variants. Results: We identied 20 mutations sites in eight patients, two heterozygous variants were identied per patient of either PDE6A or PDE6B variants, others are from CA4, OPTN, RHO, ADGRA3 variants. We identied two novel variants in PDE6A: c.1246G > A;p.(Asp416Asn) and c.1747T > A;p.(Tyr583Asn). Three novel mutations in PDE6B: c.401T > C;p.(Leu134Pro), c.2293G > C;p.(Ala765Pro) and c.1610-1612del;p. (537-538del).CA4: c.243G > A;p.(Trp81*) and RHO: c.688G>A;p.(Val230Ile) are novel variants and maybe affecting the phenotype. Among them, c.401T > C;p.(Leu134Pro) variant in PDE6B is non- pathogenic; RHO: c.688G>A;p.(Val230Ile) is conicting interpretations of pathogenicity;Other novel variants are all pathogenic. Conclusions: This study reveals novel and known variants in Chinese families with PDE6A and PDE6B mutations in autosomal recessive RP, expanding the clinical and genetic ndings of photoreceptor-specic enzyme deciencies. is conicting interpretations of pathogenicity, it maybe affecting the phenotype.The variant of ADGRA3 may not worsen the phenotype. But due to the complexity and limitations of the detection technology of gene mutation, at present, we can’t completely exclude this sequencing may be pathogenic although another pathogenic site does not detected . One limit of our study is present fundus images only in posterior pole, the periphery fundus can’t present well because of equipment incomplete. Besides, eight cases are a small sample to present the clinical and genetic features of PDE6A and PDE6B variants, we need to collect more sample to analyse in future. Overall, this study reveals novel and known mutations in Chinese families with PDE6A and PDE6B mutations in autosomal recessive RP. These ndings expand the clinical and genetic ndings of photoreceptor-specic enzyme deciencies.


Background
Retinitis pigmentosa (RP,OMIM 268000) is a heterogeneous group of inherited retinal dystrophy (IRD) characterized by night blindness, retinal degeneration with bone spicule pigmentation, constricted visual elds, and progressive disease course. The prevalence of RP is approximately 1 per 4000 persons [1].
Retinitis pigmentosa (RP) is a genetically heterogeneous disease with 65 causative genes identi ed to date. However, only approximately 60% of RP cases genetically solved to date, predicating that many novel disease-causing variants are yet to be identi ed(https://sph.uth.edu/retnet/sum-dis.htm 2021.04.28). The gene therapy and stem cell therapy for retinitis pigmentosa has a promising future, so the identi cation of novel causative variants is becoming increasingly important.
Phosphodiesterase 6(PDE6)enzyme is a heterotetrameric protein consisting of alpha(PDE6A;180071), beta (PDE6B; 180072), and 2 gamma subunits (PDE6G; 180073) [2] . Both alpha and beta subunits are required for full phosphodiesterase activity, the mechanisms by which PDE6A and PDE6B mutations lead to RP are probably similar, studies found PDE6A and PDE6B subunits are enzymatically equivalent [3], either of which is associated with recessive RP, may lead to rod death and secondarily affecting the cone photoreceptor cells [4] .
Mutations in PDE6A are found in a very low percentage of patients with RP as showed rst in a study by Huang and coworkers, suggesting a frequency of <1% [5]. Screening of about 160 patients with recessive RP in North America in a subsequent study found a frequency of mutations of approximately 3-4% [6].Mutations in PDE6B are found in a frequency of about 4% in patients from North America [1,[7][8][9].There is no statistics date about incidence rate in Chinese family. Because of the low incidence, many novel disease-causing variants are yet to be identi ed. The purpose of this study is to report the causative variants of Chinese RP families with PDE6A and PDE6B variants, expanding the clinical and genetic ndings of photoreceptor-speci c enzyme de ciencies.

Patients
Eight patients from eight unrelated families were enrolled in this retrospective study. We identi ed ve RP patients with PDE6A mutations and three with PDE6B mutations. All patients were recruited from the Department of Ophthalmology, Beijing Tongren Eye Center. Clinical diagnosis of RP was made based on clinical evaluation and electroretinograms. All medical and surgical records for the patient were reviewed. The ophthalmic examinations performed in the study patient included decimal best-corrected visual acuity (BCVA), slit lamp, funduscopy, fundus photography, visual eld testing, electroretinography (ERG), optical coherence tomography (OCT) and uorescein angiography(FFA). One hundred Chinese Han healthy individuals were selected as the control group.
Mutation screening by HEDEP Blood samples were obtained from the patients, and genomic DNA was extracted by using standard protocols. A speci c hereditary eye disease enrichment panel (HEDEP) based on targeted exome capture technology was used to collect the protein coding regions of 441 hereditary eye disease genes. Exon-enriched DNA libraries were then subjected to high-throughput sequencing using the Illumina HiSeq platform. Targeted gene enrichment, high-throughput sequencing, and data analysis were performed as described previously [10] . Brie y, exons of the target genes and adjacent portions of introns were captured by probe hybridization; enriched target genes were then sequenced with the Illumina HiSeq platform. Speci c pathogenic mutations were veri ed by Sanger sequencing.

Genetic Screening
In this study, probands P01 to P05 were PDE6A variants while P06 to P08 were PDE6B variants, all of them were heterozygous variants ,20 mutations sites were identi ed in them, including 11 missense mutations, one nonsense mutation, three splicing mutations and one delete mutation. We identi ed two novel variants in PDE6A,three novel mutations in PDE6B,CA4: c.243G > A;p.(Trp81*) in P01 and RHO: c.688G>A;p.  (Table 1).  Ophthalmoscopy showed extensive intraretinal pigment migrations extending from the mid-periphery equatorial region to the arcades in both eyes with extensive arterial attenuation, macular and peripapillary atrophy, only a small central foveal island was sparing (Fig. 2a). OCT images show high-density deposits on the surface of RPE layer in macula, residual intraretinal vacuoles and an entirely disrupted and atrophy of the retina and macular, the outer retinal structures are lost (Fig. 2b). Fluorescein angiographic show "bull's eye" macular atrophy and bonespicule hyperpigmentation blocks uorescence in large-scale of the posterior pole and spot strong uorescence (Fig. 2c). The full-eld ERG shows a decrease in rod and cone amplitude in rod response and combined rod-cone response, as well as a delayed implicit time. The 30Hz Flicker cone response also shows a decreased amplitude (Fig. 2e). In P07 (Fig. 4a-d), the proband was a 42-year old man, anterior segment examination show posterior subcapsular cataracts in both eyes,so the fundus images are not clear, attenuated vessels, and mid-peripheral bone-spicule pigmentation (Fig. 4a). OCT images show thinning of the retinal and the ellipsoid zone (EZ) is retained only in macular area (Fig. 4b).
Variants of c.1610-1612del;p.(537-538del,M2) has not been reported in RP cases previously, the deletion causes frameshift mutation, the protein structure and function of PDE6B were changed, we don't nd the variant frequency in health population in Genome Aggregation Database (gnomAD), predicted PDE6B c.1610-1612del;p.(537-538del) was the causative variants for this RP family.
In P08 (Fig. 5a-c),the proband was a 47-year old woman, fundus photographs show macular atrophy and peripapillary atrophy, attenuated vessels, and mid-peripheral bone-spicule pigmentation (Fig. 5a). Ile) to be probably damaging, disease causing, tolerated and neutra. The RHO variant has not been described in the literature, but affects a conserved amino acid residue and might also be relevant for the phenotype.The variants frequency in health population of the variant in Genome Aggregation Database (gnomAD) is 0.0039%, so, the variant is con icting interpretations of pathogenicity, it maybe affecting the phenotype. The splicing mutation of ADGRA3 just has pathogenicity while exist another mutation at the same time can lead to arRP, so it was not the causative variant for this RP family.

Clinal ndings of known variants
In P02 (Fig.6a-d), the proband was a 28-year old man who presented with 0.8 vision in both eyes. He had night blindness since infancy, fundus photographs show moderate retinal degeneration, retinal arteriolar attenuation (Fig.6a). OCT images of P02 show nearly normal thickness of macular and mild macular epiretinal membrane, conserved IS/OS line shorter than normal fundus (Fig.6b). .
In P03 (Fig.7a-f2),the proband was a 34-year old woman who presented with 0.3 best corrected visual acuity in both eyes (OD:-10.50DS/+2.00DC×90°, OS:-9.50DS/+1.25DC×75°). She had night blindness since infancy, the cataract surgery had done for both eyes, because of posterior capsular opacity, the fundus images can't presented clearly. Fundus photographs show macular atrophy and an entirely disrupted ellipsoid zone in the right eye (Fig.7a), epiretinal membrane, cystoid macular edema, outer retinoschisis,lamellar macular hole in the left eye (Fig.7b), pathological myopia maybe the reason of those phenotype. Visual elds were reduced to a small central (Fig.7c). ERGs to all stimuli were not detectable (Fig.7e).
In P05 (Fig. 8a-e), the proband was a 47-year old man who presented with 0.01 vision in his right eye and HM vision in his left eye. He had night blindness since infancy, fundus photographs show gray retinal with severe chorioretinal atrophy with bone spicule pigmentation in the area from macular to the peripheral retina, compatible with macular atrophy and structure change (Fig. 8a). OCT images show macular epiretinal membrane and vitreomacular traction and an entirely disrupted ellipsoid zone in both eyes, disappearance of the foveal depression of the right eye (Fig. 8b).Fluorescein angiographic bonespicule hyperpigmentation blocks uorescence, and the hyper uorescent spots clearly demarcate the atrophic areas (Fig. 8c).

Discussion
The phosphodiesterase 6 enzyme is involved in hydrolysis of cGMP in the photoreceptors during the transduction of light signals. PDE6A gene locates at chromosome 5, the human PDEA gene comprises 22 exons spanning approximately 45 to 50 kb and encodes for a protein containing 860 amino acids [23,24]. PDE6B gene locates at chromosome 4 and encodes for a protein containing 854 amino acids [2]. The mechanisms by which PDE6A and PDE6B mutations lead to RP are probably similar, it has been hypothesized to be due to an increased Ca 2+ in ux [25], and/or increased accumulation of cGMP [26] . The mutation of PDE6A causes retinitis pigmentosa 43, which affects the function of PDE6B [27]. Phenotypic analysis revealed no substantial differences between the two groups except for night blindness as a presenting symptom that was noted to be more prevalent in the PDE6A than PDE6B group[28].We identi ed ve RP patients with PDE6A variants and three with PDE6B variants, all of our patients complaint night blindness since the memory which consistent with previous studies that reveal that nyctalopia occurs in early childhood [9,[29][30][31][32][33].ERG presenting extinguish in most cases or only 30Hz mild reserved in previous studies [9,29,31,32,34] ,and we come to the same conclusion. The EZ width was reduced in all patients and was highly symmetric between the eyes [35][36][37], and we come to the same conclusion. When it comes to the complications of PDE6A and PDE6B variants, the most worth to pay attention to being macular abnormalities, such as vitreomacular traction, epiretinal membrane, cystoid macular edema, retinoschisis lamellar macular hole. More than half of our patients (P03,P04,P05,P06,P08)have those changes. One mechanism to explain this may be that loss of the photoreceptors elicits new glial barriers, causing Muller cells to migrate[8].
In (Leu134Pro,M1) in PDE6B just has pathogenicity while exist another mutation at the same time can lead to arRP, but exist another mutation of PDE6B c.2293G > C;p.(Ala765Pro,M2) predicted to be non-pathogenic by online tools. Analyzing the cause of this patient still along with phenotype of RP due to following reasons [40]: (1) there were larger deletions or rearrangements not detectable by Sanger sequencing; (2) there were deeper intronic mutations, which caused aberrant splicing, but were not examined in our study; (3) there were mutations in regulatory regions, which were not examined in our study; (4) there may be additional genes are responsible forRP.
In P08,variants of c.688G>A;p.(Val230Ile) in RHO gene and c.921-IG > A in ADGRA3 were noval variants, chromosome 3 that comprised the rhodopsin gene (RHO/NM_000539.3) and chromosome 4 that comprised adhesion G protein-coupled receptor A3 (ADGRA3/NM_145290.4),also named G protein-coupled receptor 125(GPR125) [41]were detected. RHO variant can causes RP, generally be autosomal dominant inheritance, but can rarely be recessive inheritance [42], pedigree P08 carries RHO c.688G>A;p.(Val230Ile) is a single site heterozygote mutation. ADGRA3 protein is a G protein-coupled receptor of unknown function, Leen Abu-Sa eh [43]found a novel splice-site mutation in a second isolated Saudi RP patient, it is associated with recessive retinitis pigmentosa; The heterozygous mutation of ADGRA3 should exist with another mutation at the same time can lead to autosomal recessive RP (arRP). So, in this arRP family, the RHO variant is con icting interpretations of pathogenicity, it maybe affecting the phenotype.The variant of ADGRA3 may not worsen the phenotype. But due to the complexity and limitations of the detection technology of gene mutation, at present, we can't completely exclude this sequencing may be pathogenic although another pathogenic site does not detected .
One limit of our study is present fundus images only in posterior pole, the periphery fundus can't present well because of equipment incomplete. Besides, eight cases are a small sample to present the clinical and genetic features of PDE6A and PDE6B variants, we need to collect more sample to analyse in future. Overall, this study reveals novel and known mutations in Chinese families with PDE6A and PDE6B mutations in autosomal recessive RP. These ndings expand the clinical and genetic ndings of photoreceptor-speci c enzyme de ciencies.

Conclusion
In

Consent for publication
Informed consent for publication is obtained from all participants or guardians on behalf of minors/child participants.

Availability of data and materials
The datasets generated and analysed during the current study are available from the corresponding author on reasonable request   Clinical observations and genetic testing in the PDE6A variant of P04. a Fundus photographs show extensive intraretinal pigment migrations and arterial attenuation in both eyes, macular and peripapillary atrophy. b OCT images show high-density deposits on the surface of RPE layer in macula, residual intraretinal vacuoles and an entirely disrupted and atrophy of the retina and macular, the outer retinal structures are lost. c Fluorescein angiographic show "bull's eye" macular atrophy (Fig. 4c). (Fig. 4e). d The pedigree of P04. e The full-eld ERG shows a decrease in rod and cone amplitude in rod response and combined rod-cone response, as well as a delayed implicit time. The 30Hz Flicker cone response also shows a decreased amplitude. f1-f3Sequence chromatogram of P04.    Clinical observations and genetic testing in the PDE6A variant of P03. a Fundus photographs of P03, the cataract surgery had done for both eyes, but because of posterior capsular opacity, the fundus images can't presented clearly, markedly severe retinal degeneration with visible atrophic choroidal vessels in the posterior retina and waxy temporal pallor of the optic disc. b OCT images show macular atrophy and an entirely disrupted ellipsoid zone in the right eye, epiretinal membrane, cystoid macular edema, outer retinoschisis lamellar macular hole in the left eye. c visual elds were reduced to a small central. d The pedigree of P03. e ERGs to all stimuli were not detectable. f1-f2 Sequence chromatogram of P03.