To report a novel hemizygous nonsense variant in the CACNA1F gene associated with congenital stationary night blindness (CSNB) in a pediatric patient, emphasizing the utility of portable electroretinography (ERG) and genetic testing in diagnosing unexplained visual impairments.
Methods
The patient, a 5-year-old male, underwent comprehensive clinical evaluation, including detailed anterior segment and fundus examinations, full-field electroretinogram (ffERG) using a RETeval™ portable device, and whole exome sequencing (WES) to elucidate the genetic basis of his visual impairment. Structural modeling of the mutated protein was performed using SWISS-MODEL and PYMOL.
Results
Best-corrected visual acuity was 0.4 logMAR bilaterally, with unremarkable anterior segment and fundus examinations. FFERG revealed significant abnormalities consistent with incomplete CSNB: severely reduced rod response in dark-adapted (DA) 0.01, negative waveform with b/a wave ratio < 1.0 in DA 3.0, and diminished cone response in light-adapted ERG. WES identified a novel pathogenic variant in the CACNA1F gene (c.1234G > T, p.E412*), inherited maternally. This variant introduces a premature stop codon at position 412, likely resulting in a truncated CACNA1F protein.
Conclusions
This case highlights the importance of comprehensive clinical assessments and genetic testing in pediatric patients with unexplained visual impairments, revealing a novel CACNA1F variant that expands our understanding of CSNB. The use of a portable ERG device proved particularly valuable in assessing retinal function in this young patient. Further investigations are warranted to elucidate the clinical implications of this novel pathogenic variant.
Lijin Wen and Yuwen Liu have equally contributed to this study.
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Introduction
Congenital stationary night blindness (CSNB) is a rare, non-progressive retinal disorder characterized by impaired night vision. This condition exhibits significant genetic and phenotypic heterogeneity, with common associated symptoms including high myopia, nystagmus, and strabismus [1]. Diagnosis primarily relies on distinctive electroretinogram (ERG) and dark adaptation test results [2]. CSNB can be categorized based on fundus appearance (normal or abnormal). The normal fundus category subdivides into the Schubert-Bornschein [3] and Riggs [4] subtypes, differentiated by full-field ERG (ffERG) findings. Additionally, Mendelian inheritance patterns further classify CSNB into autosomal dominant (adCSNB), autosomal recessive (arCSNB), and X-linked (xlCSNB) types. The Schubert-Bornschein subtype predominantly exhibits X-linked (XL) or autosomal recessive (AR) types, whereas the Riggs subtype is typically autosomal dominant (AD) [1]. The Schubert-Bornschein subtype is further divided into complete (cCSNB) and incomplete (icCSNB) types dependent on ON bipolar cells functionality and ERG results [5, 6]. CSNB with abnormal fundus includes distinct phenotypes such as fundus albipunctatus and Oguchi disease [1].
Multiple genes have been identified as causative factors for congenital stationary night blindness (CSNB) due to their pathogenic variants. These include NYX, GRM6, TRPM1, CACNA1F, GNAT1, PDE6B, RHO, SLC24A1, GPR179, LRIT3, CABP4, CACNA2D4, RDH5, RLBP1, RPE65, SAG, GRK1, and GNB3 [7]. A recent study in Saudi Arabia expanded the genetic spectrum of autosomal recessive CSNB by identifying four additional genes: RIMS2, GNB3, GUCY2D, and ABCA4 [8]. These genes are associated with syndromic cone-rod synaptic disease, ON bipolar cell dysfunction, Riggs type CSNB, and CSNB with cone-rod dystrophy, respectively. Notably, NYX and CACNA1F are primary genes associated with X-linked complete and incomplete CSNB, respectively [9]. Additionally, ongoing research continues to identify new genetic contributors to CSNB.
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The X-linked pathogenic variant in the CACNA1F gene was associated with key clinical manifestations, including hyperopia, retinal degeneration, and night blindness in the affected males. This study aims to elucidate the molecular pathology of X-linked CSNB and characterize the phenotype associated with a novel pathogenic variant in the CACNA1F gene.
Case report
A 5-year-old Chinese male was referred to our institution with a 2-year history of persistent hyperopia, amblyopia, and accommodative esotropia, which were initially diagnosed at another medical center. The patient had previously undergone amblyopia treatment; however, the visual outcomes remained suboptimal.
Upon examination, the patient's visual acuity with previous spectacles was 0.5 logMAR in each eye, improving to 0.4 logMAR bilaterally with best correction. Cycloplegic refraction revealed significant hyperopia of + 5.25 diopters (D) in the right eye and + 5.75 D in the left eye. Intraocular pressures were within normal limits bilaterally. Anterior segment examination was unremarkable in both eyes. Fundus evaluation, including ultra-widefield fundus photography (OPTOS PLC, Dunfermline, UK) and autofluorescence imaging, demonstrated no apparent abnormalities (Fig. 1A). Optical coherence tomography (OCT, Heidelberg Engineering, Inc., Heidelberg, Germany) of the macula revealed normal foveal contour and retinal thickness bilaterally (Fig. 1B). Visual evoked potentials (VEPs) were assessed with the RETI-port/scan21 (Roland Consult, Wiesbaden, Germany). For clinical assessment, two pattern check sizes (1 degree and 15 min) were used. Pattern VEPs recorded monocularly showed normal P100 peak times and amplitudes in each eye. Additionally, pattern VEPs and ffERG were recorded according to the standard International Society for Clinical Electrophysiology of Vision (ISCEV) [10]. The ffERG was carried out using the RETeval system (LKC Technologies, Inc., Gaithersburg, MD, USA). However, ffERG using the RETeval ™ portable device revealed significant abnormalities: diminished rod response in dark-adapted (DA) 0.01, negative waveform with b/a wave ratio < 1.0 in DA 3.0, and reduced cone response in light adaptation (Fig. 1C). The characteristic findings of ffEGR are consistent with the incomplete form of CSNB.
Fig. 1
Comprehensive ocular assessment of the patient
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(A) Ultra-widefield fundus photography and autofluorescence imaging demonstrating normal fundus appearance in both eyes. (B) Spectral-domain optical coherence tomography (SD-OCT) of both eyes revealing no apparent defects in retinal anatomical structure. (C) Full-field electroretinography (ffERG) results depicting light and dark adaptation responses for both eyes. light-adapted 3.0 stimulus showing severely diminished b-wave amplitudes with relatively preserved a-waves in both eyes. DA 0.01 responses demonstrating a relatively flat waveform in the right eye, with unmeasurable responses in the left eye. DA 3.0 and DA 10.0 responses with attenuated a-wave amplitudes, abolished b-wave amplitudes, resulting in a negative ERG pattern in both eyes. FFERG illustrating characteristic abnormalities associated with incomplete CSNB.
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Given the normal fundus anatomy and abnormal visual electrophysiology findings, whole exome sequencing (WES) was performed on the proband to identify potential genetic etiologies. WES analysis revealed a hemizygous nonsense pathogenic variant in the CACNA1F gene: c.1234G > T:p.E412*, which was inherited from the mother (Fig. 2) and strongly supported the diagnosis of incomplete X-linked CSNB. This variant is characterized by a substitution of guanine (G) with thymine (T) at nucleotide position 1234, leading to the alteration of the 412th codon from glutamic acid to a premature stop codon. Such a change is likely to result in a truncated and functionally defective protein product.
Fig. 2
Genetic analysis of family pedigree with identified hemizygous and heterozygous pathogenic variants
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(A) Family pedigree illustrating members affected by the pathogenic variant c.1234G > T (p.E412*) in the CACNA1F gene. (B) Sequence alignment demonstrating adenine (A) at position 49,227,011 in the CACNA1F gene for the proband and mother, indicating the presence of the pathogenic variant, while guanine (G) is observed for the father, confirming absence of the variant and suggesting maternal inheritance. (C) Sanger sequencing results for the pathogenic variant c.1234G > T (p.E412*) in family members: proband II-1 (hemizygous), father I-1 (wild type), mother I-2 (heterozygous), and sister II-2 (wild type).
To evaluate the structural impact of the p.E412* mutation, we utilized SWISS-MODEL [11] (https://swissmodel.expasy.org/) and PYMOL (https://pymol.org/) to predict and analyze the structure and function of the mutated CACNA1F protein. Sequence alignment between the wild-type and mutant proteins reveals that the mutation introduces a premature stop codon at position 412 (Fig. 3A). We generated a three-dimensional (3D) structural model of the CACNA1F protein, visualizing it through cartoon and surface representations, which emphasize the location of the E412* mutation (Fig. 3B). This mutation creates an early termination signal, resulting in a truncated protein product. The predicted model achieved a Global Model Quality Estimation (GMQE) score of 0.67, suggesting moderate reliability.
Fig. 3
Impact of CACNA1F gene mutation (c.1234G > T; p.E412) on protein structure
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The cartoon representation highlights that the E412* mutation is located within a crucial domain of the CACNA1F protein, while the surface model demonstrates the potential impact of this mutation on the protein's surface accessibility. These structural alterations provide a plausible mechanism for the loss-of-function effects associated with this mutation, as the truncated protein product may exhibit impaired stability, folding, or interactions with other essential components of the calcium channel complex.
(A) Comparison of the normal and mutated amino acid sequences. The c.1234G > T mutation introduces a premature stop codon at position 412, resulting in a truncated protein. (B) 3D structural model of the CACNA1F protein variant. The position of the p.E412* mutation is highlighted in red within a green color scheme. The disruption to the protein structure caused by the nonsense mutation p.E412* is indicated in gray. Protein models were generated using SWISS-MODEL (GMQE = 0.67).
Discussion
This study reports a novel pathogenic variant (c.1234G > T, p.E412*) in the CACNA1F gene associated with incomplete X-linked CSNB in a 5-year-old male patient. This variant, resulting in a premature stop codon, likely disrupts the normal function of the calcium channel protein encoded by CACNA1F, pivotal for retinal signal transmission. The resultant protein dysfunction manifested clinically as night blindness, and bipolar cell dysfunction in the proband, who also showed moderately high hyperopia. This pathogenic variant has not been previously reported, thereby enriching the genetic database of this disease. The identification of new pathogenic variant is crucial for improving genetic diagnosis and understanding the disease's molecular mechanisms.
The pathogenic variant in the CACNA1F gene, located on chromosome Xp11.23 and comprising 48 exons, is associated with various X-linked visual disorders, including congenital stationary night blindness, Aland Island eye disease, and cone-rod dystrophy [12, 13]. This gene, expressed in the retina’s inner nuclear, outer nuclear, and ganglion cell layers, encodes the α1-subunit (CACNA1F, Cav1.4) of L-type voltage-dependent Ca2 + channels (VDCC) [1]. Dysfunction in Cav1.4 due to these variants affects calcium ion influx and disrupts the regulation of tonic glutamate release from synaptic terminals in retinal photoreceptors and bipolar cells, thereby impairing neural signal transmission [14].
Myopia is the most common clinical sign in CSNB, accounting for 96.61% [15], while hyperopia accounts for about 22% in the previous study [9]. Our patient primarily presented with "poor vision in both eyes" and was initially diagnosed with hyperopia, amblyopia, and accommodative esotropia. Suboptimal amblyopia treatment outcomes indicate the necessity of thorough clinical evaluation and genetic testing for an accurate diagnosis. This case highlights the importance of considering CSNB in the differential diagnosis of children with unexplained visual impairment, as focusing solely on amblyopia and other common conditions may lead to misdiagnosis.
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The handheld RETeval™ device proved advantageous in evaluating retinal function in this patient. Previous studies have validated RETeval™ as a reliable and accurate tool compared to traditional full-field electroretinogram (ffERG) [16, 17]. Its noninvasiveness and convenience make it particularly suitable for children. Significant abnormalities detected included low rod response in dark adaptation (0.01), negative waveform in mixed response (3.0), and reduced cone response in light adaptation. These findings were crucial for the diagnosis of incomplete CSNB.
The identification of the c.1234G > T (E412*) pathogenic variant in the CACNA1F gene expands the known mutation spectrum linked to CSNB. CSNB is associated with mutations in 18 different genes and typically results in non-progressive night blindness [7]. Despite advances in gene discovery, some patients' causative mutations remain unidentified. The discovery of this rare mutation underscores the importance of genetic testing in patients presenting with atypical presentations. While electrophysiological examinations are essential for diagnosing hereditary retinal diseases, genetic testing provides definitive diagnostic evidence.
Conclusions
For children presenting with unexplained amblyopia and difficult-to-correct refractive errors, a comprehensive evaluation including visual electrophysiology tests (such as VEP and portable ffERG) and genetic testing is recommended to avoid misdiagnosis. Further research is warranted to elucidate the full impact of this novel pathogenic variant and to identify additional genetic variations contributing to CSNB, ultimately improving our understanding and management of this rare retinal disorder.
Declarations
Conflict of Interest
The authors declare that they have no competing interests.
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Informed consent
Informed consent was obtained from the patient’s parents.
Human rights
The study received approval from the Medical Ethics Committee of Xiang'an Hospital of Xiamen University (XAHLL2022028, March 7, 2022), in accordance with the Declaration of Helsinki.
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