Molecular Vision 2025; 31:206-219
<http://www.molvis.org/molvis/v31/206>
Received 24 January 2025 | Accepted 30 August 2025 | Published 02 September 2025
Molecular Vision 2025; 31:206-219
<http://www.molvis.org/molvis/v31/206>
Received 24 January 2025 |
Accepted 30 August 2025 |
Published 02 September 2025
Zainab Akhtar,1 Kiran Afshan,1 Yumei Li,2 Sumaira Altaf,3 Aleesha Asghar,1 Ume Sughra,4,5 Wajid Ali Khan,4 Haiba Kaul,6 Rui Chen,2 Sabika Firasat1,7
1Department of Zoology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan; 2Department of Ophthalmology, Center for Translational Vision Research, University of California, Irvine School of Medicine, CA; 3Department of Pediatric Ophthalmology and Strabismus, Al-Shifa Trust Eye Hospital, Jhelum Road, Rawalpindi, Pakistan; 4Al-Shifa School of Public Health, Pakistan Institute of Ophthalmology (PIO), Al-Shifa Trust Eye Hospital, Jhelum Road, Rawalpindi, Pakistan; 5Al-Shifa Research Centre, Pakistan Institute of Ophthalmology (PIO), Al-Shifa Trust Eye Hospital, Jhelum Road, Rawalpindi, Pakistan; 6Genetics Division, Department of Livestock Production, University of Veterinary and Animal Sciences, Ravi Campus, Pattoki, Pakistan; 7Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX
Correspondence to: Sabika Firasat; email: sabika.firasat@qau.edu.pk
Purpose: Cyclic nucleotide-gated (CNG) channels are ligand-gated ion channels that transduce light signals into electrical signals in the retinal photoreceptors. Pathogenic variants in CNG channel genes are reported to cause inherited retinal dystrophies (IRDs). The current study used targeted panel sequencing to describe the mutational spectrum of CNG channel genes in familial cases of IRDs from eight consanguineous Pakistani families.
Methods: The current study included consanguineous Pakistani families with at least two affected members. DNA was extracted from whole blood samples by the phenol-chloroform method. Two affected members from each family were initially analyzed using targeted panel sequencing of 344 known IRD genes. The pathogenicity of candidate variants was assessed using the American College of Medical Genetics and Genomics guidelines. Segregation testing was performed by Sanger sequencing.
Results: Results of eight IRD families revealed a total of four reported variants in CNGA3 (c.827A>G, c.955T>C, c.1641C>A, c.1810C>T) and three novel variants, including c.1633A>T, c.800G>T, and c.1153T>C in CNGA1, CNGA3, and CNGB3 genes, respectively, segregating in each respective family. Among disease-causing variants identified in our study cohort, 87.5% were missense. Furthermore, one of the reported missense variants (i.e., c.1641C>A in CNGA3) was segregating in two unrelated families. All identified variants were homozygous and segregated in an autosomal recessive form.
Conclusions: CNGA3 was the most frequently mutated gene in our study cohort. Only the c.1641C>A variant of CNGA3 was repeated in two families, showing genetic diversity. The identification of three novel pathogenic variants in CNG channel genes in the present study reaffirms the allelic and genetic heterogeneity of IRDs in the Pakistani population.
Retinal cyclic nucleotide-gated (CNG) channels are ligand-gated ion channels that belong to the superfamily of the pore-loop cation channel [1]. They are found on the plasma membranes of the outer segment of photoreceptor cells and mediate visual phototransduction by binding cyclic guanosine monophosphate (cGMP) [2–4]. In the absence of light, dark-adapted photoreceptors maintain a high level of cytosolic cGMP, which permits CNG channels to stay open [5,6]. When light activates rhodopsin, a visual pigment in rod photoreceptors, it triggers a cascade of enzymatic reactions that ultimately lead to the breakdown of cGMP by phosphodiesterase. This causes a decrease in cGMP levels, which leads to the closure of ion channels, preventing Na+ and Ca2+ from entering and a change in membrane potential [7,8]. The light-induced CNG channel closure lowers the cytosolic Ca2+ concentration that, in turn, modulates the sensitivity of the CNG channel and activates guanylate cyclase to resynthesize cGMP [1,6,9]. A similar Ca2+-dependent CNG channel pathway operates in the cone photoreceptors, but their molecular constituent is somewhat different from its rod counterpart [6].
Cone and rod photoreceptors express distinct CNG channels encoded by homologous genes [10]. In mammals, six homologous genes (CNGA1, CNGA2, CNGA3, CNGA4, CNGB1, and CNGB3) encode for structurally similar A and B subunits that assemble in discrete combinations into cell type–specific heterotetrameric complexes [11]. Each heterotetrameric CNG channel is made by three A subunits and one B subunit (CNGA1 and CNGB1 in rods but CNGA3 and CNGB3 in cones) [2]. Membrane proteins encoded by each of the six CNG channel genes consist of six helical transmembrane segments (S1–S6), a channel core comprising a reentrant pore loop between S5 and S6, and cytosolic N- and C-terminals. The pore domain is formed by S5, S6, and the intervening reentrant pore loop [6,11,12]. The intracellular cGMP-binding domain is located at the C-terminus, which is joined to S6 by the C-linker [10].
Four of the CNG channel genes (CNGA1, CNGA3, CNGB1, CNGB3) are linked to inherited retinal dystrophies (IRDs) [10]. Mutations in the rod-specific channel genes (CNGA1 or CNGB1) cause rod dystrophies/autosomal recessive retinitis pigmentosa (RP)/congenital stationary night blindness [13–15]. A literature search shows that mutations in CNGA1 and CNGB1 account for 1% to 8% and 4% of autosomal recessive RP cases, respectively. Cone-specific channel gene (CNGA3 or CNGB3) mutations cause cone dystrophy or achromatopsia/cone-rod dystrophy [16–18]. Collectively, mutations in CNGA3 and CNGB3 are responsible for 69% of achromatopsia cases [19,20].
Due to genetic diversity in IRDs, molecular testing is crucial for a definitive diagnosis and patient management. In an ongoing effort to study the mutational spectrum of IRDs in consanguineous Pakistani families [21,22], here we report pathogenic variants in CNG channel genes identified from familial cases belonging to eight unrelated families along with a summary of previously reported CNG channel gene mutations from cases of Pakistani origin.
The approval of the presented research work was taken from the Bio-Ethical Review Committee of the Faculty of Biologic Sciences, Quaid-i-Azam University Islamabad, Pakistan, and the Ethical Review Committee, Al-Shifa Trust, Rawalpindi, Pakistan. Clinical assessment of families was done by ophthalmologists at Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan, to confirm the diagnosis of IRD phenotype in each family. Clinical assessments include detailed family and medical history, physical examination, fundoscopy, slit-lamp exam, and visual acuity testing. Families were recruited following the principles of the Declaration of Helsinki for molecular genetic investigation. Written informed consent was obtained from enrolled families before drawing 3 to 4 ml of venous blood from participating affected and control individuals. Genomic DNA extraction and quantification were performed at the Department of Zoology, Quaid-i-Azam University, Islamabad, Pakistan, as per our previously reported method [21].
DNA of two affected individuals from each enrolled IRD family was used for capture panel sequencing at Baylor College of Medicine, Houston, Texas. Exome-enriched genomic libraries were prepared using the KAPA HyperPrep Kit (Roche, Basel, Switzerland) following the manufacturer’s protocol, then combined for targeted enrichment of a panel of 344 known genes of IRD-related genes, as described in our previous article [21] with the SureSelect Target Enrichment System for the Illumina Platform (Agilent, Santa Clara, CA) [23]. Using a Novaseq 6000 (Illumina, San Diego, CA), captured DNA was measured and sequenced. As mentioned in our earlier publications, variant calling, data alignment, and filtering were performed at the Functional Genomics Core at Baylor College of Medicine in the United States [21–23]. Variant annotation and filtration were performed according to the American College of Medical Genetics and Genomics recommendations. The HGMD, ClinVar, and LOVD databases were searched for and used to identify previously known pathogenic variants. Several in silico tools were used to assess novel variations for possible effects on protein function, including REVEL v1.3 [24]. Splice site variations, nonsense, and frameshift variants were categorized as probable loss-of-function alleles. Sequence conservation and bioinformatics predictions were used to assess missense variants.
Primer3 web-based resource was used to design primers to validate each pathogenic variant. Genomic DNA of available affected and unaffected members of each family were amplified for the suspected gene(s) and subjected to Sanger sequencing.
The eight families reported in this study (selected from 72 inherited retinal dystrophies segregating Pakistani families) were recruited from Al-Shifa trust eye hospital, Rawalpindi, Pakistan. Among these enrolled families, four (RD001, RD028, RD053, and RP125) were Punjabi, two (RD021 and RD025) were Pashtun, and two (RD004 and RD031) were of Kashmiri ethnicity. Clinical data of the affected individuals of these families recorded at enrollment are detailed in Table 1. Comprehensive interviews of elders of each family revealed ethnicity, age of onset, symptoms, and complete family history for drawing pedigrees (Figure 1, Appendix 1 and Appendix 2). All enrolled families had more than one affected individuals. Among 27 affected members, 11 were males and 16 were females. All the enrolled probands had congenital onset of the disease phenotype. Symptoms including decreased visual acuity, color blindness, photophobia, nyctalopia, and hemeralopia of the proband of each family are listed in Table 1. Enrolled affected cases had nystagmus eyes except RD031 (IV.III) and RD053 (IV.III). Refractive error and hyperopia were present in the affected members of RD004 (IV.III), RD021 (IV.IV), RD028 (VI.IV), and RD031 (IV.II). Other ocular and phenotypic features, including squint and astigmatism, were recorded for patient of RD021 (IV.I). Affected cases of RD053 had only nyctalopia and hemeralopia. Fundus examination of family RD021 (IV.I; Appendix 3) showed small hyperopic changes with a deep cup, a cup-to-disc ratio of 0.4 to 0.5, bull’s-eye maculopathy, and periphery unrecordable pigmentary changes.
Targeted panel sequencing followed by Sanger sequencing validation confirmed segregation of seven pathogenic variants in CNG channel genes (i.e., CNGA1, CNGA3, and CNGB3 with IRD phenotype in eight screened families). There was one stop gain and six missense variants. From the identified variants, three are novel to the current study, and four have been previously reported (Table 2 and Table 3). All identified pathogenic variants were homozygous, segregated with the disease phenotype in an autosomal recessive form. Detailed pedigree of each with segregation of the identified novel/reported variant is given in Figure 1 and Appendix 1 and Appendix 2, respectively. All the identified variants were classified as per guidelines from the American College of Medical Genetics and Genomics, and their details are provided in Table 2.
Among the three novel variants identified in this study, a missense variant, c.1153T>C, causing p.(Trp385Arg) in CNGB3, was observed in a Pashtun family (RD021) with two affected siblings (Table 2). Both children belonged to unaffected consanguineous parents and had congenital disease onset with symptoms positive for the cone-rod dystrophy phenotype. Both affected children also had squinting eyes. This observed variant caused the substitution of tryptophan with arginine. Segregation was tested in one affected and two controls of the family (Figure 1B and Figure 2B). This variant is absent from the gnomAD (v2.1.1) database.
A missense variant c.1633A>T of the CNGA1 gene was observed in a Punjabi family (i.e., RD053 segregating recessively with the RP phenotype; Table 2). Genotyping of an affected member and two unaffected members confirmed the segregation of identified variants (Figure 1C and Figure 2C). The reported gnomAD (v2.1.1) allele frequency for this variant is 0.00002406.
All missense substitutions of CNGA3 were identified in exon 8. A novel missense variant c.800G>T in the CNGA3 gene was found in a Kashmiri family (RD004) having four affected females (Table 2). Segregation of this pathogenic variant in an autosomal recessive manner was confirmed by genotyping three affected females and one unaffected female (Figure 1A and Figure 2A). This variant substitutes glycine for valine at position 267 of the encoded protein. This variant is absent from the gnomAD (v2.1.1) database.
Among three previously reported missense variants detected in the CNGA3 gene, one (c.1641C>A) segregated in two unrelated Punjabi consanguineous families, RD001 and RD028, with the same clinical phenotype (Table 2). The variant p.(Phe547Leu) caused by a single base transition in exon 8 was segregating in an autosomal recessive manner in eight affected individuals (Appendix 1 and Appendix 4). The family RD001 had five affected cases in two successive generations while RD028 had three cases in a single generation. Cases were homozygous for the variant from a heterozygous carrier parent. This variant is classified as pathogenic or likely pathogenic in ClinVar with a minor allele frequency of 0.0001592 in gnomAD (v2.1.1). A consanguineous Pashtun family RD025 with two affected sisters was homozygous for the c.955T>C variant, which results in p.(Cys319Arg; Table 2). Other ocular findings observed in this family were hemeralopia and nyctalopia. Segregation analysis was performed by genotyping two affected and four unaffected individuals (Appendix 1 and Appendix 4). This missense variant is reported as a pathogenic/likely pathogenic in ClinVar, with a minor allele frequency of 0.00001767 in gnomAD (v2.1.1). Another previously reported missense variant identified in RP125 was c.827A>G causing p.(Asn276Ser) in the CNGA3 gene. The segregation of this variant was confirmed by analyzing four unaffected females and one affected female (Appendix 1 and Appendix 4). This reported variant is absent from the gnomAD (v2.1.1) database.
In a Kashmiri family (RD031), a stop-gain variant c.1810C>T of the CNGA3 gene causing premature protein termination (i.e., p.(Gln604*)) was segregating in a homozygous recessive manner with disease phenotype (Table 2). Segregation of this variant was confirmed in five affected and three unaffected members, supporting variant pathogenicity (Appendix 2 and Appendix 4), with a gnomAD (v2.1.1) frequency of 0.000003993. Codes met and curated modified classification of previously reported and novel variants based on the data of the present study are listed in Table 2.
Understanding the genetic spectrum of individuals with IRDs is imperative for the development of future suitable treatment strategies due to the involvement of multiple genes for the same disease etiology or the same gene disease-causing variant(s) resulting in altered phenotype(s). In the current study, seven disease-causing variants in three CNG channel-associated genes, CNGA1, CNGA3, and CNGB3, were identified in eight consanguineous Pakistani families. Previous studies have indicated that the prevalence of CNGA3 and CNGB3 disease-causing variants varies geographically. The CNGA3 variants are more common in Asian populations, whereas CNGB3 variants are more prevalent in Europe and the United States [25]. This observation is consistent with our current findings, since among eight families analyzed in this study, six were segregating pathogenic variants in the CNGA3 gene (75%; Table 2). A list of 16 CNGA3 variants given in the Table 3, including previously published variants from the Pakistani population and those identified in this study, shows that all except two are missense alleles (Table 3). Interestingly, we identified a previously reported missense variant (i.e., c.1641C>A) in CNGA3 segregating in two unrelated consanguineous Punjabi families, RD001 and RD028 (Table 2). Previously, this variant was reported as a homozygous and heterozygous achromatopsia (ACHM)/cone dystrophy (CD)-causing allele from different populations across the globe [17,26–30], including Pakistan [31,32] (Table 3).
Two families, RD025 and RP125, were identified with two reported missense variants p.(Cys319Arg) and p.(Asn276Ser) in the CNGA3 gene, respectively (Table 2). Previously, both of these variants p.(Cys319Arg) and p.(Asn276Ser) were reported for the first time as homozygous recessive disease-causing alleles from the Pakistani population [4,18,31,33]. Later on, the variant p.(Cys319Arg) was also reported as a heterozygous allele causing the ACHM phenotype in a Pakistani family in a compound heterozygous manner along with another heterozygous allele, p.(Arg436Trp) [15]. However, in our study, all affected individuals were homozygous for the p.(Cys319Arg) variant, segregating ACHM/cone-rod dystrophy (CRD) phenotype (Table 1 and Table 2).
The homozygous c.1810C>T variant causing p.(Gln604*) in family RD031 is the only stop-gain variant in CNGA3 identified in this study. This pathogenic variant is reported as likely pathogenic in ClinVar. Variant identification by targeted panel sequencing followed by Sanger sequencing in five affected and three unaffected members supports the variant pathogenicity (Appendix 2 and Appendix 4).
Among the three novel missense variants identified in the current study, one was c.800G>T in the CNGA3 gene (Table 2). All three affected sisters with the p.(Gly267Val) mutation had reduced visual acuity, defective color vision, severe photophobia, and nystagmus, with complaints of nyctalopia. This variant substitutes glycine for valine at position 267 in the protein transcript, causing an alteration in protein structure and function. The second novel variant c.1153T>C, causing p.(Trp385Arg), was observed in the CNGB3 (cyclic nucleotide-gated channel, beta-3) gene, which encodes the beta subunit of a CNG ion channel playing a role in phototransduction [26]. This variant was segregating with disease phenotype and had additional ocular abnormalities in family RD021. With the identification of this novel CNGB3 missense variant, the total number of disease-causing variants identified to date from Pakistan has increased to six. Among these six, only two are missense variants, whereas the remaining four are null alleles (Table 3). In the present study, the only identified missense variant in the CNGA1 (cyclic nucleotide-gated channel, alpha-1) gene was c.1633A>T segregated with an autosomal recessive RP phenotype. Previously, homozygous and compound heterozygous mutations in the CNGA1 gene have been reported to cause RP in cases from East Asian and European populations [14,34–37]. Our literature search indicated that previously, just two missense mutations in the CNGA1 gene were identified from cases of Pakistani origin, highlighting the rare incidence of CNGA1 mutations in our population (Table 3). Identification of a novel homozygous missense variant, segregating with the RP phenotype in an affected case of the RD053 family, supports the previous findings of the association of CNGA1 with rod dystrophies/RP. All these novel variants are found highly conserved in diverse species by using Clustal Omega and Weblogo3 (Figure 3A–D).
Our literature searches to catalog all the disease-causing variants described until December 2024 in the CNG channel genes from Pakistan and identification of the three novel disease-causing variants described in the present study reveal their mutation spectrum and contribution to the total genetic load of autosomal recessive IRDs in Pakistan (Table 2 and Table 3). Furthermore, given the difference in CNGA1 (in rod) and CNGA3 (in cone) expression in photoreceptors, we observed a significant difference in the phenotype of patients. The RD053 patient segregated CNGA1 and had an RP phenotype in which rod photoreceptors were affected first, whereas in cases of families with mutations in the CNGA3 gene, cone-rod dystrophy was detected. Interestingly, our study cohort included 72 families with IRD, among which we identified disease-causing variants in CNG channel genes (CNGA1, CNGA3, and CNGB3) in eight families (11.1%), but we did not identify any mutation in the CNGB1 gene. The main limitation in our study explaining genetic variants to phenotypic diversity is the unavailability of electroretinography reports due to the lack of this facility in the collaborating hospital. Our study highlights the need to invest in better diagnostic equipment in Pakistan to capture more comprehensive clinical data for future research. Nevertheless, this study contributes to the genetic spectrum of the CNG channel-associated phenotypes within the Pakistani population and provides practical implication of genetic screening in the specific diagnosis of IRD subtypes.
Appendix 2. Pedigree of family RD031 with a stop gain reported variant in CNGA3 gene.
Appendix 3. Fundus photographs of left and right eye of an affected member of family RD021 (IV-I).
We thank patients and their families for their participation in this study. Author Contribution: Rui Chen is a co-corresponding author and email addresses of both corresponding authors are sabika.firasat@qau.edu.pk and ruic20@hs.uci.edu. Author Contribution: Sabika Firasat, Kiran Afshan and Rui Chen contributed to the conceptualization and design. Ume Sughra, Wajid Ali Khan and Sumaira Altaf performed clinical evaluation of patients. Zainab Akhtar, Aleesha Asghar and Haiba Kaul enrolled families and collected data. Zainab Akhtar, Yumei Li and Sabika Firasat performed experiments and data analysis. Zainab Akhtar, Yumei Li, Kiran Afshan, Haiba Kaul, Sabika Firasat and Rui Chen prepared the first draft of the manuscript. All authors read and approved the content and submission of the final manuscript. Conflict of interest: We have no conflict of interest. Funding: This study was financially supported (in part) by the University Research Fund (URF) from Quaid-i-Azam University, Islamabad, Pakistan. This work is also supported by grants from the National Eye Institute [R01EY022356, R01EY018571, R01EY002520, P30EY010572, R01EY09076, R01EY030499]; Retinal Research Foundation; NIH shared instrument grant [S10OD023469]. The authors acknowledge support to the Gavin Herbert Eye Institute at the University of California, Irvine from an unrestricted grant from Research to Prevent Blindness and from NIH grant P30EY034070