Introduction: Retinitis pigmentosa (RP), a heterogeneous inherited retinal disorder causing gradual vision loss, affects over 1 million people worldwide. Pathogenic variants in CNGA1 and CNGB1 genes, respectively, accounting for 1% and 4% of cases, impact the cyclic nucleotide-gated channel in rod photoreceptor cells. The aim of this study was to describe and compare genotypic and clinical characteristics of a cohort of patients with CNGA1- or CNGB1-related RP and to explore potential genotype-phenotype correlations. Methods: The following data from patients with CNGA1- or CNGB1-related RP, followed in five Italian inherited retinal degenerations services, were retrospectively collected: genetic variants in CNGA1 and CNGB1, best-corrected visual acuity (BCVA), ellipsoid zone (EZ) width, fundus photographs, and short-wavelength fundus autofluorescence (SW-AF) images. Comparisons and correlation analyses were performed by first dividing the cohort in two groups according to the gene responsible for the disease (CNGA1 and CNGB1 groups). In parallel, the whole cohort of RP patients was divided into two other groups, according to the expected impact of the variants at protein level (low and high group). Results: In total, 29 patients were recruited, 11 with CNGA1- and 18 with CNGB1-related RP. In both CNGA1 and CNGB1, 5 novel variants in CNGA1 and 5 in CNGB1 were found. BCVA was comparable between CNGA1 and CNGB1 groups, as well as between low and high groups. CNGA1 group had a larger mean EZ width compared to CNGB1 group, albeit not statistically significant, while EZ width did not differ between low and high groups A statistically significant correlation between EZ width and BCVA as well as between EZ width and age were observed in the whole cohort of RP patients. Fundus photographs of all patients in the cohort showed classic RP pattern, and in SW-AF images an hyperautofluorescent ring was observed in 14/21 patients. Conclusion: Rod CNG channel-associated RP was demonstrated to be a slowly progressive disease in both CNGA1- and CNGB1-related forms, making it an ideal candidate for gene augmentation therapies.

Retinitis pigmentosa (RP), first described in 1853, is a clinically and genetically heterogeneous group of inherited retinal disorders that causes a gradual loss of vision [1, 2]. RP has a prevalence of approximately 1:4,000, affecting more than 1 million people worldwide [3, 4], and is most frequently inherited in an autosomal recessive manner (arRP) [3]. The disease is characterized by a progressive retinal degeneration caused by loss of photoreceptors cells, with rods being affected first. Hence, the first symptom of RP is generally nyctalopia, which is followed by a gradual narrowing of the visual fields, to finally result in loss of visual acuity once the cones start to degenerate [1, 3]. Fundus examination typically shows intraretinal pigmentation (referred to as bone-spicule deposits because of their shape), vascular narrowing, and optic nerve pallor [1‒3]. Since the discovery of the first gene responsible for RP in 1990 [5], mutations in about 70 genes have been implicated in non-syndromic RP (https://web.sph.uth.edu/retnet/sum-dis.htm#A-genes) [6]. Each of these disease-causing genes corresponds to a gene-specific subtype of RP. For some genes, the phenotype is superimposable between different subtypes, whereas for others the clinical manifestations and disease progression can be extremely variable [7].

Pathogenic variants in CNGA1 and CNGB1 genes, respectively, account for 1% and 4% of autosomal recessive manner cases [8, 9]. These genes encode the alpha and beta subunits that assemble the rod cyclic nucleotide-gated (CNG) channel, responsible for the “dark current.” In fact, CNG channels in the outer segment of rods mediate the influx of Na+ and Ca2+, when maintained in the open state by a high intracellular concentration of guanosine 3′,5′-cyclic monophosphate (cGMP) in the dark. This allows rods to depolarize, which in turn promotes synaptic glutamate release [10‒12].

To date, significant advances in the genetics of RP have been matched by significant progress in the development of novel strategies for its treatment [13, 14], among which one of the most promising seems to be gene augmentation. Gene augmentation therapy-based approaches aim at restoring wild type expression in target cells, using a virus-based vector [15]. Therefore, an important prerequisite of these approaches is the presence of living target cells in order to be effective. In two recent preclinical studies, gene augmentation therapy has also been proposed for patients with pathogenic variants in CNGB1. In particular, in two animal models for CNGB1-related RP, gene augmentation therapy was shown to rescue the phenotype, improve rod function, and reduce retinal degeneration over time [16‒19]. Gene augmentation therapy is being proposed for many genetic retinal degenerations, and it led to the first FDA-approved therapy, Luxturna, for the treatment RPE65-Associated Retinal Dystrophy [20‒23].

Since gene therapy for RP patients is only successful when there are still sufficient functional rods remaining to support continued cone survival [21], these promising initial gene therapy results must be supported by evidence that there is a substantial preservation of photoreceptors in patients with CNGA1- or CNGB1-associated RP, thereby offering a relatively large therapeutic window. To date, studies describing clinical characteristics of CNGB1-related RP patients are few [24‒27], and data about CNGA1-related RP natural history is limited. Therefore, the aim of this study was to describe and compare genotypic and clinical characteristics of a group of RP patients with pathogenic variants in CNGA1 and CNGB1 and to explore potential genotype-phenotype correlations.

Patient Selection

A total of 29 patients with a clinical and molecular diagnosis of rod CNG channel-associated RP, belonging to 26 unrelated families, were included in this study. Their phenotype was caused by pathogenic variants either CNGA1 or CNGB1, in, respectively, 11 and 18 cases. All patients were recruited and underwent detailed clinical examinations in one of the five Italian hospitals involved in the study: ASST Santi Paolo e Carlo Hospital (Milan), AOU Careggi (Florence), Bambino Gesù IRCCS Children’s Hospital (Rome), Agostino Gemelli Hospital (Rome), IRCCS Fondazione Bietti (Rome). Before genetic testing, all patients had undergone pretest counselling and had been informed about the significance of genetic testing. Written informed consent, in compliance with the Declaration of Helsinki, was obtained from all the patients. The study protocol was approved by the Local Ethical Committee of Azienda Sanitaria dell’Alto Adige, Italy (Approval No. 132-2020).

The patients’ clinical data were collected retrospectively from the clinical records. Collected data included best-corrected visual acuity (BCVA), spectral-domain optical coherence tomography, short-wavelength fundus autofluorescence (SW-AF) images, and ophthalmic fundus photographs. Ellipsoid zone (EZ) width was measured as previously described by our group [28]. SW-AF images were classified into two groups, based on the presence or absence of hyperautofluorescent (hyperAF) ring within the limit of the 30° lens of the Heidelberg Spectralis HRA and OCT machine.

Genetic Testing

Genomic DNA was isolated from peripheral blood or saliva using the SaMag Blood DNA Extraction Kit) (Sacace Biotechnologies, Como, Italy) according to the manufacturer’s instructions. Following the extraction of DNA, the samples were subjected to next-generation sequencing (NGS) on an Illumina MiSeq personal sequencer (Illumina, San Diego, CA, USA). The DNA probe set was designed to capture the coding exons and flanking regions of each gene of the panel using the Twist Custom Panel Design Technology (Twist Custom Panel kit; Twist Bioscience, South San Francisco, CA, USA) that included CNGB1 (NM_001297.5) and CNGA1 (NM_001379270.1). The complete list of genes included in the panel has been published elsewhere [9]. An average of 0.6 Mbases (±0.1) per sample were obtained by sequencing, resulting in mean coverage of targeted bases of 65× (±9.2) per sample. On average, 98.5% (±0.15%) of all bases were covered at least 10×, 97% (±1%) of bases had coverage >25×. The in-house developed PipeMAGI bioinformatics pipeline was used for the analysis of the raw sequencing data, resulting in the identification and annotation of sequence variants [29]. The analysis of copy number variants (CNVs) was performed using NGS data as previously described elsewhere [30]. The pathogenicity of the identified variants was assessed according to the American College of Medical Genetics and Genomics (ACMG) standard and guidelines [31] with the help of the online tool VarSome (https://varsome.com/) [32, 33].

Next, the identified variants in CNGA1 and CNGB1 were divided into two groups, based on their expected impact (low/high) at the protein level. More specifically, missense and intronic variants outside of canonical splice sites were considered to have a low impact at the protein level, while copy number variants, nonsense and frameshift variants, and variants in canonical splice sites (±1, ±2) were considered to have a high impact.

When possible, segregation studies were performed on the probands’ family members (both healthy and affected) to establish the phase of the variants in order to enhance their classification. Furthermore, regardless of the clarified allelic arrangement, patients were designated as high if both variants were classified as having a high impact at the protein level according to the aforementioned criteria. Conversely, patients were categorized as Low if at least one of the variants was classified as having a low impact (Table 1).

Table 1.

Genetic variants of CNGA1 and CNGB1 identified in RP patients

IDFamilySexAge at examinationSegregation analysisGeneAllelic stateNucleotide change INucleotide change IIAmino acid change IAmino acid change IIVarSome verdict IVarSome verdict IIHigh/lowReference IReference II
Fam-1 61 Yes CNGA1 Comp-HET c.1327dup c.1528C>T p.(Thr443Asnfs*3) p.(Arg510*) High [9[34
Fam-2 59 Yes CNGA1 HOM c.1528C>T c.1528C>T p.(Arg510*) p.(Arg510*) High [34[34
Fam-3 80 Yes CNGA1 HOM c.1743_1746del c.1743_1746del p.(Thr582Serfs*17) p.(Thr582Serfs*17) High [35[35
Fam-4 70 Yes CNGA1 HOM c.438–2A>G c.438–2A>G p.(?) p.(?) High [9[9
Fam-5 74 Yes CNGA1 HOM c.1240A>G c.1240A>G p.(Lys414Glu) p.(Lys414Glu) VUS VUS Low 
Fam-6 66 No CNGA1 HOM c.614_617del c.614_617del p.(Ile205Thrfs*3) p.(Ile205Thrfs*3) LP LP High 
Fam-7 85 Yes CNGA1 HOM c.154_165delinsTTCTGAGGATGAAGACAGTG c.154_165delinsTTCTGAGGATGAAGACAGTG p.(Glu52Phefs*2) p.(Glu52Phefs*2) LP LP High 
Fam-8 46 No CNGA1 HOM del ex 4-5-6 del ex 4-5-6 p.(?) p.(?) LP LP High 
Fam-9 43 Yes CNGA1 HOM c.1477C>T c.1477C>T p.(Pro493Ser) p.(Pro493Ser) VUS VUS Low 
10 Fam-9 39 Yes CNGA1 HOM c.1477C>T c.1477C>T p.(Pro493Ser) p.(Pro493Ser) VUS VUS Low 
11 Fam-10 43 No CNGA1 HOM c.1873C>T c.1873C>T p.(Arg625*) p.(Arg625*) High [36[36
12 Fam-11 61 CNGB1 Unknown c.2629G>A c.413-1G>A p.(Gly877Arg) p.(?) LP Low [9[37
13 Fam-12 46 Yes CNGB1 Comp-HET c.1957+2T>G c.1333G>T p.(?) p.(Glu445*) High [9[9
14 Fam-13 45 Yes CNGB1 HOM c.1256_1260del c.1256_1260del p.(Glu419Glyfs*10) p.(Glu419Glyfs*10) LP LP High 
15 Fam-14 52 Yes CNGB1 HOM c.2762_2765del c.2762_2765del p.(Tyr921Cysfs*15) p.(Tyr921Cysfs*15) High [38[38
16 Fam-15 60 Yes CNGB1 Comp-HET c.827_834del c.2957A>T p.(Ile276Thrfs*4) p.(Asn986Ile) LP Low [9[39
17 Fam-15 54 Yes CNGB1 Comp-HET c.827_834del c.2957A>T p.(Ile276Thrfs*4) p.(Asn986Ile) LP Low [9[39
18 Fam-16 56 Yes CNGB1 Comp-HET c.1949C>T c.2544dup p.(Pro650Leu) p.(Leu849Alafs*3) LP Low [9[24
19 Fam-17 30 Yes CNGB1 Comp-HET c.2957A>T c.1975T>C p.(Asn986Ile) p.(Trp659Arg) LP LP Low [39
20 Fam-17 36 Yes CNGB1 Comp-HET c.2957A>T c.1975T>C p.(Asn986Ile) p.(Trp659Arg) LP LP Low [39
21 Fam-18 53 No CNGB1 Unknown c.2528_2541delinsACCATCACCATCA c.2957A>T p.(Leu843Hisfs*21) p.(Asn986Ile) LP LP Low [39
22 Fam-19 55 No CNGB1 HOM del ex 29 del ex 29 p.(?) p.(?) LP LP High 
23 Fam-20 50 No CNGB1 HOM c.2296T>C c.2296T>C p.(Cys766Arg) p.(Cys766Arg) LP LP Low [9[9
24 Fam-21 84 No CNGB1 Unknown c.413-1G>A c.1917G>A p.(?) p.(Trp639*) High [40[25
25 Fam-22 77 No CNGB1 Unknown c.217+5G>A c.2764_2765del p.(?) p.(Glu922Valfs*11) VUS LP Low [25
26 Fam-23 35 No CNGB1 HOM c.413-1G>A c.413-1G>A p.(?) p.(?) High [40[40
27 Fam-24 88 No CNGB1 HOM c.217+5G>A c.217+5G>A p.(?) p.(?) VUS VUS Low [25[25
28 Fam-25 62 No CNGB1 Unknown c.875-5_891dup c.2957A>T p.(Gly298Cysfs*13) p.(Asn986Ile) LP LP Low [25[39
29 Fam-26 64 Yes CNGB1 HOM c.2762_2765del c.2762_2765del p.(Tyr921Cysfs*15) p.(Tyr921Cysfs*15) High [38[38
IDFamilySexAge at examinationSegregation analysisGeneAllelic stateNucleotide change INucleotide change IIAmino acid change IAmino acid change IIVarSome verdict IVarSome verdict IIHigh/lowReference IReference II
Fam-1 61 Yes CNGA1 Comp-HET c.1327dup c.1528C>T p.(Thr443Asnfs*3) p.(Arg510*) High [9[34
Fam-2 59 Yes CNGA1 HOM c.1528C>T c.1528C>T p.(Arg510*) p.(Arg510*) High [34[34
Fam-3 80 Yes CNGA1 HOM c.1743_1746del c.1743_1746del p.(Thr582Serfs*17) p.(Thr582Serfs*17) High [35[35
Fam-4 70 Yes CNGA1 HOM c.438–2A>G c.438–2A>G p.(?) p.(?) High [9[9
Fam-5 74 Yes CNGA1 HOM c.1240A>G c.1240A>G p.(Lys414Glu) p.(Lys414Glu) VUS VUS Low 
Fam-6 66 No CNGA1 HOM c.614_617del c.614_617del p.(Ile205Thrfs*3) p.(Ile205Thrfs*3) LP LP High 
Fam-7 85 Yes CNGA1 HOM c.154_165delinsTTCTGAGGATGAAGACAGTG c.154_165delinsTTCTGAGGATGAAGACAGTG p.(Glu52Phefs*2) p.(Glu52Phefs*2) LP LP High 
Fam-8 46 No CNGA1 HOM del ex 4-5-6 del ex 4-5-6 p.(?) p.(?) LP LP High 
Fam-9 43 Yes CNGA1 HOM c.1477C>T c.1477C>T p.(Pro493Ser) p.(Pro493Ser) VUS VUS Low 
10 Fam-9 39 Yes CNGA1 HOM c.1477C>T c.1477C>T p.(Pro493Ser) p.(Pro493Ser) VUS VUS Low 
11 Fam-10 43 No CNGA1 HOM c.1873C>T c.1873C>T p.(Arg625*) p.(Arg625*) High [36[36
12 Fam-11 61 CNGB1 Unknown c.2629G>A c.413-1G>A p.(Gly877Arg) p.(?) LP Low [9[37
13 Fam-12 46 Yes CNGB1 Comp-HET c.1957+2T>G c.1333G>T p.(?) p.(Glu445*) High [9[9
14 Fam-13 45 Yes CNGB1 HOM c.1256_1260del c.1256_1260del p.(Glu419Glyfs*10) p.(Glu419Glyfs*10) LP LP High 
15 Fam-14 52 Yes CNGB1 HOM c.2762_2765del c.2762_2765del p.(Tyr921Cysfs*15) p.(Tyr921Cysfs*15) High [38[38
16 Fam-15 60 Yes CNGB1 Comp-HET c.827_834del c.2957A>T p.(Ile276Thrfs*4) p.(Asn986Ile) LP Low [9[39
17 Fam-15 54 Yes CNGB1 Comp-HET c.827_834del c.2957A>T p.(Ile276Thrfs*4) p.(Asn986Ile) LP Low [9[39
18 Fam-16 56 Yes CNGB1 Comp-HET c.1949C>T c.2544dup p.(Pro650Leu) p.(Leu849Alafs*3) LP Low [9[24
19 Fam-17 30 Yes CNGB1 Comp-HET c.2957A>T c.1975T>C p.(Asn986Ile) p.(Trp659Arg) LP LP Low [39
20 Fam-17 36 Yes CNGB1 Comp-HET c.2957A>T c.1975T>C p.(Asn986Ile) p.(Trp659Arg) LP LP Low [39
21 Fam-18 53 No CNGB1 Unknown c.2528_2541delinsACCATCACCATCA c.2957A>T p.(Leu843Hisfs*21) p.(Asn986Ile) LP LP Low [39
22 Fam-19 55 No CNGB1 HOM del ex 29 del ex 29 p.(?) p.(?) LP LP High 
23 Fam-20 50 No CNGB1 HOM c.2296T>C c.2296T>C p.(Cys766Arg) p.(Cys766Arg) LP LP Low [9[9
24 Fam-21 84 No CNGB1 Unknown c.413-1G>A c.1917G>A p.(?) p.(Trp639*) High [40[25
25 Fam-22 77 No CNGB1 Unknown c.217+5G>A c.2764_2765del p.(?) p.(Glu922Valfs*11) VUS LP Low [25
26 Fam-23 35 No CNGB1 HOM c.413-1G>A c.413-1G>A p.(?) p.(?) High [40[40
27 Fam-24 88 No CNGB1 HOM c.217+5G>A c.217+5G>A p.(?) p.(?) VUS VUS Low [25[25
28 Fam-25 62 No CNGB1 Unknown c.875-5_891dup c.2957A>T p.(Gly298Cysfs*13) p.(Asn986Ile) LP LP Low [25[39
29 Fam-26 64 Yes CNGB1 HOM c.2762_2765del c.2762_2765del p.(Tyr921Cysfs*15) p.(Tyr921Cysfs*15) High [38[38

New variants are reported in bold. Patients were considered resolved when their allelic status was compound-heterozygous (Comp-HET) or homozygous (HOM) and the two identified genetic variants were pathogenic (P) or likely pathogenic (LP), and not variants of uncertain significance (VUS) by VarSome.

F, female; M, male; Fam, family.

Statistical Analysis

Analyses were performed on 1 eye per patient. For each patient, metrics of only the worst eye were used for analysis. The worst eye was defined as the one with lower BCVA or (in case of identical BCVA) shorter EZ width. First, we compared BCVA and EZ width dividing patients in two groups according to the gene responsible for disease; then we did the same comparison but dividing the whole cohort of RP patients in two groups based on the expected impact of the variants at the protein level, as reported in the previous paragraph.

Data normality was tested according to Kolmogorov-Smirnov. Continuous variables were described as mean ± standard deviations, while categorical variables by using frequency. Covariance analysis was applied to the dataset by appointing visual acuity as dependent variable and EZ width, disease, age, and allele status as covariates. In case of statistically significant results, the differences between groups were inspected using unpaired two-sample t test (continuous data) or χ2 (categorical data). We also tested Pearson correlations between variables of interest. Depending on the values of the correlation coefficient (r) correlation was considered almost perfect (0.81–0.99), substantial (0.61–0.80), moderate (0.41–0.60), fair (0.21–0.40), or slight (0.01–0.20). The level of significance used during the statistical analyses was set at p < 0.05. The analyses were performed using IBM SPSS (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0.; IBM Corp., Armonk, NY, USA).

Genetic analysis of the whole RP cohort (n = 29) resulted in the identification of 27 genetic variants in CNGA1 and CNGB1 (Table 1). Among the identified variants, 5 genetic variants in CNGA1 and 5 genetic variants in CNGB1 were not reported in literature. Among the novel variants, 8 were classified as likely pathogenic (LP) and 2 were classified as variants of unknown significance according to ACMG criteria.

Familial segregation analysis was performed for 15 out of 29 probands. Overall, 20 out of the 29 probands were considered genetically resolved by carrying LP and/or pathogenic (P) variants, either in homozygous or in compound heterozygous state (confirmed through segregation analysis).

Considering the whole cohort of RP patients, 8 patients had disease-causing variants in CNGA1 and 12 in CNGB1. For both groups, demographic and clinical characteristics are reported in Table 2. For patients in the CNGA1 group, mean BCVA was similar to patients in the CNGB1 group (0.65 ± 0.25 vs. 0.66 ± 0.37). Even EZ width was not significantly different between CNGA1 group and CNGB1 group: the mean value of patients in the former was 4,057 ± 1,301 and in the latter 3,395 ± 1,942 (p = 0.38).

Table 2.

Clinical characteristics of the whole cohort of RP patients, divided either by gene (CNGA1 and CNGB1 group) or by impact (low/high)

CharacteristicPatients (n = 29)CNGA1 (n = 11)CNGB1 (n = 18)Low (n = 14)High (n = 15)
Age at the visit, years 
 Mean (min-max)±SD 50 (25–83)±16 52 (28–77)±17 48 (25–83)±16 48 (25–76)±17 52 (32–83)±16 
Ethnicity 
 Italian 24 15 10 14 
 Albanian 
 Ethiopian 
 Egyptian 
Gender, n (%) 
 Female 15 (52) 3 (27) 12 (67) 8 (57) 7 (47) 
 Male 14 (48) 8 (73) 6 (33) 6 (43) 8 (53) 
BCVA, decimal Snellen 
 Mean±SD 0.66±0.33 0.65±0.25 0.66±0.37 0.64±0.35 0.67±0.31 
EZ, µm 
 Mean±SD 3,584±1,777 (n = 21) 4,057±1,300 (n = 6) 3,395±1,942 (n = 15) 3,252±1,995 (n = 8) 3,789±1,679 (n = 13) 
SW-AF 
 HyperAF ring 14 11 
 Absence of HyperAF ring 
 Unknown 
CharacteristicPatients (n = 29)CNGA1 (n = 11)CNGB1 (n = 18)Low (n = 14)High (n = 15)
Age at the visit, years 
 Mean (min-max)±SD 50 (25–83)±16 52 (28–77)±17 48 (25–83)±16 48 (25–76)±17 52 (32–83)±16 
Ethnicity 
 Italian 24 15 10 14 
 Albanian 
 Ethiopian 
 Egyptian 
Gender, n (%) 
 Female 15 (52) 3 (27) 12 (67) 8 (57) 7 (47) 
 Male 14 (48) 8 (73) 6 (33) 6 (43) 8 (53) 
BCVA, decimal Snellen 
 Mean±SD 0.66±0.33 0.65±0.25 0.66±0.37 0.64±0.35 0.67±0.31 
EZ, µm 
 Mean±SD 3,584±1,777 (n = 21) 4,057±1,300 (n = 6) 3,395±1,942 (n = 15) 3,252±1,995 (n = 8) 3,789±1,679 (n = 13) 
SW-AF 
 HyperAF ring 14 11 
 Absence of HyperAF ring 
 Unknown 

Fundus photographs were available for 7 patients in the CNGA1 group and 15 in the CNGB1 group. All subjects in both groups showed a typical and symmetrical form of RP, mainly involving the periphery and with different degrees of the classic RP triad: waxy pallor of the optic disc, attenuation of retinal vessels, and diffuse bone-spicule pigmentation (shown in Fig. 1).

Fig. 1.

Fundus photographs (a, b), SW-AF (c, d), and OCT (e, f) of 2 patients from our cohort. Images on the left (a, c, e) belong to patient 13, who carries two variants in CNGB1 (Table 1). Images on the right (b, d, f) belong to patient 4, who carries two variants in CNGA1 (Table 1). Fundus photographs (a, b) show different degrees of retinal involvement. SW-AF (c, d) show different patterns: preserved central autofluorescence surrounded by an hyperautofluorescent ring (c) and central increase of autofluorescence with hypoautofluorescent macular lesion (d). SD-OCT (e, f) show EZ width segmentation: limits (white line) were considered where the hyper-reflective band declined to zero. SD-OCT, spectral-domain optical coherence tomography.

Fig. 1.

Fundus photographs (a, b), SW-AF (c, d), and OCT (e, f) of 2 patients from our cohort. Images on the left (a, c, e) belong to patient 13, who carries two variants in CNGB1 (Table 1). Images on the right (b, d, f) belong to patient 4, who carries two variants in CNGA1 (Table 1). Fundus photographs (a, b) show different degrees of retinal involvement. SW-AF (c, d) show different patterns: preserved central autofluorescence surrounded by an hyperautofluorescent ring (c) and central increase of autofluorescence with hypoautofluorescent macular lesion (d). SD-OCT (e, f) show EZ width segmentation: limits (white line) were considered where the hyper-reflective band declined to zero. SD-OCT, spectral-domain optical coherence tomography.

Close modal

SW-AF images were available for 5 patients in the CNGA1 group and 16 in the CNGB1 group. HyperAF ring was detectable at the posterior pole or beyond the arcades in 3 out of 5 and 11 out of 16 patients in the CNGA1 and CNGB1 group, respectively. Among the other patients, hyperAF ring was not visible at SW-AF imaging and altered macular autofluorescence was detected (shown in Fig. 1).

Considering the whole cohort of RP patients and following the criteria described above, 14 patients were classified in the low impact group and 15 in the high impact group; demographic and clinical characteristics of these two groups are reported in Table 2. Among the 18 CNGB1-RP patients, 11 (61%) were classified in the low impact group and 7 (39%) in the high impact group. Among the 11 CNGA1-RP patients, 3 (27%) were classified in the low impact group and 8 (73%) in the high impact group. BCVA and EZ width values were not significantly different between low and high impact groups: mean BCVA was 0.69 (±0.34) in the low impact group and 0.63 (±0.32) in the high impact group. Mean EZ width was 3,638 micron (±1,769) in the high impact group and 3,496 micron (±1,908) in the low impact group. Considering the whole cohort of RP patients, the correlations between EZ and BCVA and between EZ and age were substantial (r = 0.62 and r = −0.62, respectively, shown in Fig. 2).

Fig. 2.

Correlation between EZ width and BCVA (a) and EZ width and age (b) in the whole cohort of RP patients.

Fig. 2.

Correlation between EZ width and BCVA (a) and EZ width and age (b) in the whole cohort of RP patients.

Close modal

Pathogenic variants in CNGA1 gene cause RP49 (OMIM 613756), while variants in CNGB1 gene are responsible for RP45 (OMIM 613767). Our study aimed to genetically and clinically investigate a cohort of patients, followed in five Italian IRDs reference centers, reporting some newly identified CNGA1 and CNGB1 genetic variants, presenting clinical data of RP49 and RP45 phenotypes, and comparing the natural course of these two subtypes of RP by means of morphological, demographic, and psycho-physical measurements. CNGA1 and CNGB1 are expressed in rods and form CNG channels by combining three alpha 1 subunits (encoded by CNGA1 gene) and 1 beta subunit (encoded by CNGB1 gene). In parallel, the two subunits from the cone CNG channels are encoded by CNGA3 and CNGB3. Notwithstanding similarity in the structure of alfa and beta subunits, absence or downregulation of one of the two subunits results in photoreceptors degeneration [11].

In the 29 probands analyzed in this study, 5 genetic variants in CNGA1 and 5 in CNGB1, not previously reported in literature, were identified. Among them, 7 are expected to have a highly detrimental effect on the proteins (CNV and frameshift variants). Moreover, within our study cohort, we observed that 8 out of 11 patients with CNGA1 variants carried at least one high impact variant (73%), while this was the case for 7 out of 18 patients with CNGB1 variants (39%). These findings align with the existing literature [12], which describes that a significant number of CNGA1 variants cause deletions of key functional domains. Conversely, the majority of CNGB1 variants lead to minor deletions or single amino acid substitutions, with milder effects on related proteins [12]. Among these genetic variants, one of the most frequently observed in populations from different continents is the c.2957A>T p.(Asn986Ile) [25]. Notably, this variant was identified in 6 out of 18 patients (33% of cases) with CNGB1 variants in our cohort, further underlining its common occurrence.

The discovery of new variants is of fundamental importance for a correct genetic characterization of patients in the era of precision medicine. From this point of view, it is equally important to have increasingly exhaustive clinical data on the natural history of the disease, in order to identify the therapeutic window as well as to be able to identify the endpoints that can best measure the course of the disease and the possible efficacy of therapeutic strategies. Clinical data of CNGB1-related RP were already described in some previously published studies [24‒27]: overall, it is recognized as a slowly progressive disease with central vision sparing. Regarding BCVA, our data are consistent with previous findings: Jackson et al. [26] reported a mean VA of 0.5 decimal in a cohort of 33 patients with mild changes over a follow-up of about 4.5 years; Hull et al. [24] of 0.8 decimal in a small cohort of 10 patients; Nassisi et al. [25] reported that 75% of their cohort of 32 patients retained a BCVA ≥0.5 decimal. Likewise, 15 out of 18 patients in our study (83%) had a BCVA superior to 0.3 decimal and the mean BCVA was 0.66 decimal Snellen.

Although there are no reported data on the mean EZ width in a cohort of RP45 patients, we can compare our data with those of our previous paper where we evaluated the EZ width at OCT in patients with syndromic or non-syndromic forms of RP related to the USH2A gene [41], with autosomal recessive transmission pattern. In patients with CNGB1-related RP, the mean ± SD EZ width was 3,395 ± 1,942 microns with a mean age of 48 years, in contrast to the non-syndromic and syndromic more aggressive USH2A-related forms, where it was 1,969 ± 1,411 (mean age 52 years) and 1,306 ± 1,246 (mean age 44 years) microns, respectively. Our cohort’s data confirm, by the analysis of the EZ width, which objectively reflects disease progression [42‒46] a slower evolving form of RP than USH2A-related RP. SW-AF is another important tool to study photoreceptor integrity and disease progression over time [47‒49] since it is recognized to be a less variable compared to functional assessments like BCVA and visual field [50]. HyperAF ring marks the transition between healthy and atrophic outer retina: its presence in RP patients is an indicator of preserved central function [47]. In order to characterize our cohort and understand whether the hyperAF ring may be a good parameter for disease follow-up, as well as an endpoint for future therapy studies, we assessed its presence/absence. In agreement with literature data on both RP45 patients and other forms of RP, about 70% of our cohort showed the presence of hyperAF sign [25, 51, 52].

To the best of our knowledge, clinical data on patients with CNGA1-related RP is limited, thus for the first time we described clinically, as well as genetically, a cohort of patients with RP49, and again the data delineate a slowly progressing form. Indeed, there were no significant differences in terms of BCVA and EZ width compared to RP45. Interestingly, none of the patients in CNGA1 group had low vision: even the oldest patient (85 years old) retained a BCVA of 0.3 decimal in the worst eye. By contrast, 3 out of 18 CNGB1 patients presented a BCVA of count fingers or worse. While age might play a role (2/3 are 66 and 76 years old), the presence of a 39-year-old patient suggests that other genetic or environmental factors may play a role in determining the faster progression in some CNGB1 cases [53].

The presence of the hyperAF ring in 3 out of 5 patients (60%), albeit in a very small number of cases, seems to further support the hypothesis that even in CNGA1 patients the macular region is preserved over a wide time window. Given the similarity between RP49 and RP45, the action on the same target (CNG channel), and the low number of patients included in the two groups, we also decided to divide the entire cohort based on the expected impact (low/high) of the variants at the protein level, in order to evaluate potential genotype-phenotype correlations. In contrast to the results found in patients with USH2A-related retinal dystrophy [41] and in line with observations reported on previously described cohorts of patients with RP45 [25], we found no possible associations: the BCVA and EZ width did not significantly differ between the two groups. In addition, the 2 out of 3 patients with low vision described above belonged to the low class, further confirming this observation.

Moreover, considering the entire RP cohort studied, our study reiterates that EZ width substantially correlates with both age and BCVA, as reported in the literature [54], making it a meaningful measurement to follow up disease progression of CNG-related phenotypes. The main limitation of this study is the small cohort of patients, which does not permit us to draw definite conclusions; moreover, it relies on retrospective data. However, despite the small cohort, this study was able to identify 10 new genetic variants in CNGA1 and CNGB1 genes, confirm literature data on RP45, and describe for the first time the clinical features of RP49, which, like CNGB1-related RP, turns out to be a slowly progressing form and therefore an ideal candidate for gene augmentation therapies. Studies on larger cohorts will be needed to provide useful information in the scope of personalized therapy. Indeed, coupling genetic data with clinical data and identifying reliable endpoints of the disease course is essential to plan gene therapy trials.

The study protocol was approved by the Local Ethical Committee of Azienda Sanitaria dell’Alto Adige, Italy (Approval No. 132-2020). Written informed consent was obtained from participants to participate in the study.

The authors have no conflicts of interest to declare.

This research was funded by Regione Toscana in the scope of EJPRD JTC 2020 TreatRP.

Conceptualization, methodology, visualization, and : L.C. and P.E.M.; formal analysis: G.B. and P.E.M.; investigation: G.I., L.Z. P.F., V.D.R., V.M., D.G., B.F., G.P., S.M, and E.G.; resources: L.C., G.I., L.Z. P.F., V.D.R., V.M., D.G., B.F., G.P. S.M., E.G., and M.B.; data curation: G.B. and P.E.M.; writing – original draft preparation: L.C., G.B, and P.E.M.; writing – review and editing: G.I., L.Z. P.F., V.D.R., V.M., D.G., B.F., G.P., S.M., E.G., M.B., and L.R.; supervision: L.C. and M.B.; project administration: L.C., M.B., and L.R.; funding acquisition: V.M. and M.B. All authors have read and agreed to the published version of the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author and to Doctor Leonardo Colombo (leonardo.colombo.82@gmail.com).

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