Mutational Profile and Retinal Phenotypes of PCARE-Related Cone-Rod Dystrophies in a Mexican Cohort

Purpose The aim of the study is to describe the genotype and phenotype of a Mexican cohort with PCARE-related retinal disease. Methods The study included 14 patients from 11 unrelated pedigrees with retinal dystrophies who were demonstrated to carry biallelic pathogenic variants in PCARE. Visual assessment methods included best corrected visual acuity, color fundus photography, Goldmann visual field test, kinetic perimetry, dark/light adapted chromatic perimetry, full-field electroretinography, autofluorescence imaging, and spectral domain-optical coherence tomography imaging. Genetic screening was performed either by gene panel sequencing or by exome sequencing. Results According to the results of multimodal imaging and functional tests, all 14 patients were diagnosed with cone-rod dystrophy. Six different PCARE pathogenic alleles were identified in our cohort, including three novel mutations: c.3048_3049del (p.Tyr1016∗), c.3314_3315del (p.Ser1105∗), and c.551A > G (p.His184Arg). Notably, alleles p.His184Arg, p.Arg613∗, and p.Arg984∗ were present in 18 of the 22 (82%) PCARE alleles from probands in our cohort. Conclusion Our work expands the PCARE mutational profile by identifying three novel pathogenic variants causing retinal dystrophy. While phenotypic variations occurred among patients, a cone-rod dystrophy pattern was observed in all affected individuals.


Introduction
Inherited retinal degenerations (IRDs) are a heterogeneous group of genetic disorders characterized by progressive dysfunction of neuroretinal cells and/or the retinal pigment epithelium.IRDs have a combined prevalence of approximately one case per 2,000 individuals [1] and are considered the most common cause of blindness in the working-age population [2].IRDs are a paradigmatic example of diseases with phenotypic and genotypic variabilities, as their age of onset, severity of clinical manifestations, and rate of visual loss are considerably variable among individuals and as mutations in over 280 genes can result in the disease [3,4].IRD-related genes participate in a myriad of cellular processes that are essential for the structural and functional integrity of retinal tissue [5].Genetic screening of individuals with IRDs is a crucial procedure for accurate diagnosis, prognosis, and management of patients, and it has been tremendously improved in recent years by the widespread incorporation of next-generation sequencing (NGS) techniques [6,7].
Pathogenic variants in PCARE, a gene located at 2p23.2 and previously known as C2orf71, has been recently demonstrated in IRDs [8,9].Recessive loss-of-function variants in this gene have been reported in subjects with diferent retinal phenotypes involving both rods and cones, namely retinitis pigmentosa (RP) and cone-rod dystrophy (CRD) [10][11][12][13].PCARE-related IRD is associated with wide variation in terms of age of onset and progression rate of retinal damage with vision loss typically beginning in the second or third decade of life; about half of the patients also present with nyctalopia at the time of consultation, and vision can be preserved until the ffth or sixth decade [11].Fundus appearance is particularly variable, ranging from early-onset maculopathy to changes resembling retinitis pigmentosa [11].Te prevalence of PCARE-related IRD is currently unknown, but it is expected to vary from 1% as observed in a French cohort [14] to 15% in a Swiss population [10].
To date, more than 30 PCARE pathogenic variants have been recognized in patients sufering from diverse retinal phenotypes, most of them from European and Asian ethnicities [15,16].Te majority of these variants correspond to single nucleotide variants or indels that generate premature stop codons or frameshifts [17].
Te aim of this study was to describe the phenotypes and disease-causing variants in a cohort of PCARE-related IRD patients from Mexico.Our results add to the knowledge of the clinical and molecular spectrum of retinal dysfunction due to PCARE mutations.

Study Population.
Te study corresponded to a retrospective and descriptive case series comprising 14 afected patients with retinal disease who were demonstrated to carry causative variants in PCARE; all included patients were of Mexican origin.Te study was approved by the Institutional Review Board of the Institute of Ophthalmology "Conde de Valenciana" in Mexico City.All procedures adhered to the tenets of the Declaration of Helsinki, and written informed consent was obtained from the participants.

Clinical Data.
Medical records were reviewed to collect clinical data and participants underwent a complete eye examination, including visual acuity testing, ultrawide fundus photography, Goldmann visual feld kinetic perimetry, dark/light adapted chromatic perimetry (Metrovision, MonCvONE, France), autofuorescence imaging (FAF), spectral domain-optical coherence tomography (SD-OCT) (Spectralis; Heidelberg Engineering, Heidelberg, Germany), and full-feld electroretinography (fERG) (Metrovision, MonPackONE, France).Mean defcits in chromatic perimetry scores were calculated using an internal database of values from normal Mexican population.Te ERG protocol complied with the standards of the International Society for Clinical Electrophysiology of Vision.

Genetic Analysis. PCAREdisease-causing mutations
were identifed by either gene panel (patients #1-9, 14) or exome sequencing (ES) (patients #10-13).Briefy, genomic DNA (gDNA) was extracted from peripheral blood leukocytes using the QIAamp DNA Blood Mini Kit (Qiagen, Germany), following the manufacturer's protocol.In patients #1-9, and 14 gDNA was enriched for targeted regions using a hybridization-based protocol and sequenced using Illumina technology.Sequence analysis and deletion/duplication testing were performed on 298 genes included in the Invitae Inherited Retinal Disorders Panel (Invitae, San Francisco, CA).Target regions were sequenced with ≥50x depth, and reads were aligned to the GRCh37/Hg19 reference sequence.In patients #10-13, ES was performed at 3Billion, Inc. (Seoul, South Korea).DNA library preparation was performed using the IDT xGen Exome Research Panel v2.0 kit (Integrated DNA Technologies, Coralville, Iowa, USA), and sequenced on NovaSeq 6000 (Illumina, San Diego, CA, USA).Te mean depth-of-coverage was 140x with a minimum of 98.5% of the targeted region covered at 30x.Te base call (BCL) sequence fles generated by NovaSeq 6000 were converted and demultiplexed to FASTQ fles using bcl2fastq v2.20.0.422.Sequence reads in the FASTQ fles were aligned to the human reference genome (GRCh37/ hg19) using BWA-mem 0.7.17 to generate BAM fles.BAM fles were processed following the GATK best practices (GATK v.3.8) for variant calling to generate VCF fles.Exome sequencing data annotation and variant fltration were performed using the Franklin platform (Genoox, Palo Alto, CA).Te designation of pathogenic or likely pathogenic variants was performed according to the American College of Genetics and Genomics (ACMG) guidelines.In silico analysis and visualization of residue conservation and position at the protein level were performed using Jalview (jalview.org)and ConSurf-DB (consurfdb.tau.ac.il), respectively, in relevant missense variants.Variants of clinical signifcance were confrmed and segregated in families by Sanger sequencing.Primers sequences and PCR conditions are available on request.

Patients' Summary.
A total of 14 patients pertaining to 11 unrelated families sufering from PCARE-related retinal dystrophies were included in the analysis.Tables 1 and 2 present their demographic and clinical data; two families (II and V) with more than one afected subject were ascertained.Te cohort included eight males (57%) and six females.Te mean age of symptoms onset was 17.2 years (±11.05,range 5-36), and the mean age upon clinical examination was 36 years (±11.80,range 16-54).Family VI reported a history of consanguinity, while family IX reported endogamy.

Ocular Phenotypes.
All patients had clinical data from at least one ophthalmologic examination, which revealed a tendency towards symmetric afectation (Tables 1 and 2).Te symptoms at the onset of the disease were nyctalopia (8/ 14, 57%), vision loss (3/14, 21%), photophobia (2/14, 14%), and photopsia (1/14, 7%).Visual acuity at examination 2 Journal of Ophthalmology     Journal of Ophthalmology ranged from 0.09 logMAR to light perception, with 10 out of 13 available patients having in both eyes a logMAR value of 0.50 or worse.Funduscopic common fndings included a tessellated appearance, hypopigmented dotting, optic disc pallor, and vessel attenuation (Figure 1).Atrophic maculopathy was observed in six patients (ID# 2, 3, 6, 11, 12, and 14).Chromatic perimetry (available in four patients, #7-9, 11) showed a reduction in the response of both rod and cone systems; photopic mean defcits ranged from 12.63 dB to 20.14 dB, and the scotopic mean defcits ranged from 20.08 dB to 41.9 dB.Five patients (ID# 2, 3, 6, 12, and 13) with low visual acuity or poor fxation were unable to perform a chromatic perimetry test, so a full-feld stimulus threshold test (FST) was completed instead.Te FST showed a reduced response under scotopic conditions and a more reduced response under photopic conditions in all these patients (Table 3).Goldmann perimetry results were available in seven patients.Preservation of a central island of vision was noted in four patients (ID# 2, 3, 8, 10); additionally, patient #8 had a nasal island of vision in the periphery.In the youngest patient (ID# 7), the visual feld was normal using V4e stimulus, while a perifoveal relative scotoma with an augmented peripapillary scotoma was found in the OD using the I4e stimulus.In 11 patients in whom FAF test was performed, the most frequent fndings were hypo-AF with a nummular or mottling pattern in the periphery (ID# 2, 3, 6-14); hyper-AF foveal dot (ID# 2, 3, 7-13); hypo-AF in the central macular area (ID# 2, 3, 6, 9-14); peripapillary hypo-AF (ID# 2, 3, 6, 9, 12, 13); and hyper-AF macular ring surrounding the mentioned hypo-AF macular area (ID# 3, 7-10, 14) (Figure 1).All ten patients who underwent full-feld ERG had an abolished response in scotopic, mesopic, and photopic conditions, with the exception of patient #7 who had a subnormal scotopic and mesopic response in addition to an abolished photopic response (see supplementary Table (available here)).

Journal of Ophthalmology
SD-OCT images, available in 11 patients, demonstrated loss of outer retinal layers to diferent degrees in all of them, preservation of the ellipsoid zone (EZ) in four (ID# 7, 8, 10, 13), and severely atrophic damage with loss of retinal architecture in fve patients (ID# 2, 3, 6, 11, and 12).Outer retinal tubulations (ORT) appeared in three patients (ID# 2, 3, 12) (Figure 1).According to multimodal imaging and functional assessment, all 14 patients were diagnosed with CRD.

Genetic Findings.
All afected individuals carried biallelic PCARE pathogenic alleles, including a total of 6 distinct gene variants (Table 4).Pathogenic alleles included four nonsense (two novel: p.Tyr1016 * and p.Ser1105 * ), one frameshift, and one novel missense variant (p.His184Arg).Notably, three variants (p.His184Arg, p.Arg613 * , and p.Arg984 * ) were present in 18 of the 22 PCARE alleles from the 11 probands (82%).Segregation analysis confrmed compound heterozygosity in four probands while a homozygous status was confrmed in the other seven.Based on the American College of Medical Genetics and Genomics criteria for variant assessment, all fve identifed null variants were classifed as pathogenic, while the single identifed novel missense variant (p.His184Arg) was classifed as likely pathogenic.Of note, the p.His184Arg variant afects a highly conserved residue (GERP: 5.52) (Figure 2) and has an extremely low frequency (gnomAD 0.0004%), and in silico molecular analysis with diferent tools predicts a deleterious efect (SIFT: 0, PolyPhen-2: 1, VARITY: 0.92).

Discussion
In the present study, we describe in depth the clinical and genetic features of 14 patients, from 11 diferent pedigrees, with biallelic disease-causing PCARE variants.To our knowledge, this is one of the largest cohorts of PCARErelated IRDs reported to date and the frst to include Latin American patients, a highly underrepresented population in published IRDs cohorts.
PCARE stands for photoreceptor cilium actin regulator, which is the name given to the protein product.As its name indicates, it is specifcally expressed in the retina [8], and there is evidence showing its localization in the connecting cilium of photoreceptor cells where it participates, along with other proteins (e.g., WASF3), in actin dynamics for the expansion of the ciliary membrane, an important process for outer segment development and homeostasis [18][19][20].
In our cohort, the clinical fndings showed a generalized photoreceptor disorder with early macular involvement, compatible with a CRD pattern.Te clinical diagnosis of CRD was established mainly based on the results of chromatic perimetry, since there was a relatively better response under scotopic conditions in all the patients.Early macular atrophy and prominent cone-system deterioration have been mentioned in several reported cases of PCARE retinopathy, usually co-occurring with manifestations of rodsystem deterioration [9][10][11]14].
Te descriptive data obtained from this cohort show, in general terms, a variable expression of the disease, including a variable age at onset ranging from the frst to the fourth decade of life, and heterogeneity in the severity of visual symptoms.Indeed, nyctalopia as the frst reported symptom in some patients is unexpected for a CRD diagnosis, and this could be the result of early and severe damage in regions with a high density of rods, including some regions of the macula, along with cone damage.All of our patients presented visual loss to some degree, progressing to light perception vision after the fourth decade of life; as expected, older individuals exhibited worse visual acuity in our cohort.Notably, clinical variation appeared to be less marked among subjects pertaining to the same family than among patients from unrelated pedigrees.
ORTs were observed on OCT images from three patients in our cohort.Tese are tubular retinal structures formed in the outer nuclear layer by reorganization of photoreceptors 6 Journal of Ophthalmology  in diferent stages of degeneration, Müller cells and RPE cells [21].Tese structures have been associated with a variety of retinal conditions, such as age-related macular degeneration, diabetic retinopathy, choroidopathies, and retinal dystrophies [22,23].Our observation of ORTs in PCARE-related IRD is consistent with previous reports [11,15], and notably, the three subjects presented the ORTs in both eyes.In two cases, asymmetric ORTs in terms of their size were observed and, remarkably, in both cases, the eye with the larger ORTs presented better visual acuity (Table 1).Interestingly, a previously reported PCARE-related IRD case series described three patients with ORTs who appeared to have better visual acuity than the rest of the cohort [11].Although the clinical signifcance of ORTs is controversial, some authors have recognized their potential use as clinical biomarkers of prognosis [24,25].Te genetic characterization of our cohort identifed a total of six diferent PCARE pathogenic variants, including three novel mutations.Although all the included families were apparently unrelated, certain PCARE variants had a noticeably higher frequency than others.Interestingly, two of these common variants, p.Arg613 * and p.Arg984 * , have been reported in patients of Korean [11] and French [14] ethnicities, respectively, suggesting that these could be recurrent gene variations.In contrast, the p.His184Arg missense variant, observed in 4 out of 22 probands' alleles, had not been previously reported.While a possible explanation for this high frequency is a founder mutation efect in our population, additional haplotype analyses are required to confrm this hypothesis.
In accordance with previous reports, our results indicate that the majority of IRD-related PCARE variants are predicted null variants.Tus, fve diferent null variants and a single missense variant were characterized in our cohort, with all six having a predicted loss-of-function efect.Of note, fve out of six variants identifed here introduce premature termination codons (PTCs), and according to the canonical rules known for nonsense-mediated mRNA decay activation [26], all of them are predicted to activate the mechanism leading to transcript degradation.On the other hand, mutation modeling shows that the novel p.His184Arg variant afects a highly conserved residue of the PCARE protein (Figure 2), located in a likely structured region corresponding to a helical coiled coil domain, which is predicted to have an important role in the function of the protein [18,19].Previously characterized pathogenic PCARE missense variants as p.Ile201Phe [8] and p.Cys599Arg [27] also afect relatively conserved residues.Even so, a clear genotype-phenotype correlation was not established in our cohort.
In conclusion, we report one of the largest cohorts of PCARE-related retinal dystrophy, and the frst that includes Latino American population.Our results support that CRD is the main phenotype related to PCARE defects and confrm  8 Journal of Ophthalmology the association of ORT to the disease, an OCT fnding that could serve as a clinical biomarker of the disorder.We also expand the mutational spectrum of PCARE with the report of three novel disease-causing variants. Photophobia

Figure 1 :
Figure 1: Retinal images of patients.Te left column shows ultra-wide fundus photography, the middle column shows autofuorescence imaging, and the right column shows SD-OCT images.Every row corresponds to a diferent patient.

Table 1 :
Demographic, clinical features, and genetic variants of the PCARE-related Mexican cohort.