Abstract
Background
Inherited retinal degenerations are a group of genetically heterogeneous disorders characterized by progressive photoreceptor degeneration. Despite the widespread use of targeted gene panels and exome sequencing, many cases remain genetically unresolved because structural and other complex variants may be missed. CEP290 is a major cause of early-onset retinal degeneration, and although most pathogenic variants are sequence-level changes, structural rearrangements have increasingly been recognized through genome-wide sequencing approaches. This study aimed to identify the genetic cause in a child with early-onset retinal degeneration and to characterize the molecular mechanism of the underlying variant.
Methods
A 4-year-old boy with presumed rod-cone dystrophy underwent comprehensive clinical evaluation and trio whole-genome sequencing. Compound heterozygous CEP290 variants, consisting of a maternally inherited splice-site variant and a paternally inherited large intragenic duplication, were identified and evaluated. The duplication breakpoint was subsequently characterized by structural variant analysis, gap-PCR, and Sanger sequencing, followed by analysis of breakpoint-associated microhomology, GC content, and repeat elements. Variants were interpreted according to ACMG/AMP and ClinGen recommendations, as appropriate.
Results
Trio-WGS detected compound heterozygous CEP290 variants: a maternal splice-site variant c.6012–2 A > G and a paternal exons 31–53 tandem duplication (chr12:88050034–88089159dup). Gap-PCR and Sanger sequencing validated this rearrangement and defined a 7-bp junction microhomology (AAATTCT). Breakpoint analysis showed low GC and abundant repeats, supporting FoSTeS/MMBIR as the causal replicative mechanism. This duplication is predicted to alter CEP290 transcripts, triggering frameshift, premature termination and C-terminal domain loss.
Conclusions
To our knowledge, this study represents the first breakpoint-resolved characterization of a pathogenic CEP290 tandem duplication encompassing exons 31–53 identified in compound heterozygosity with a pathogenic splice-site variant in a patient with early-onset retinal degeneration. The duplication is predicted to disrupt the CEP290 coding sequence, resulting in a frameshift, premature termination codon, and loss of the C-terminal functional domain. These findings expand the spectrum of pathogenic structural variants in CEP290 and underscore the value of whole-genome sequencing and breakpoint-level analysis for resolving genetically unexplained inherited retinal disorders.
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s12920-026-02435-1.
Keywords: CEP290, Large duplication, Copy number variation, Early-onset retinal degeneration
Introduction
Inherited retinal degeneration (IRD) refers to a group of genetic disorders characterized by progressive degeneration of retinal photoreceptors, including retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt disease, and cone/cone-rod dystrophies (CORD). IRDs display marked clinical and genetic heterogeneity and may involve extraocular manifestations such as auditory, neurological, skeletal, renal, cardiac, and hepatic abnormalities [1].
Inherited retinal degenerations are among the most genetically heterogeneous Mendelian disorders, with more than 280 disease-causing genes implicated across diverse inheritance patterns [2]. While targeted gene panels and whole-exome sequencing (WES) are routinely used in clinical practice, with WES remaining the most widely established platform for routine molecular diagnosis, a substantial proportion of patients remain genetically unsolved. This diagnostic gap largely stems from technical limitations in detecting complex structural variants, deep intronic variants, and variants located in difficult-to-sequence genomic regions [3, 4]. Whole-genome sequencing (WGS) provides more comprehensive and uniform genomic coverage than targeted panel sequencing or WES and is particularly valuable for resolving previously unsolved cases by detecting structural variants, deep intronic variants, and other complex genomic rearrangements [5–7].
CEP290-associated retinal degeneration (CEP290-IRD) accounts for approximately 15%-20% of Leber congenital amaurosis (LCA) cases, and the gene is a leading genetic cause of childhood blindness [8, 9]. Typical pathological features of CEP290-IRD include disorganization of photoreceptor outer segments, early selective loss of mid-peripheral rods with relative preservation of macular cones, and intact optic nerve and occipital cortex despite reduced photoreceptor input [9, 10]. These features indicate a therapeutic window in which residual cones can be targeted to restore vision. Therefore, elucidating the pathogenic mechanisms of CEP290 defects and developing corresponding therapeutic strategies are of major clinical and scientific significance.
The CEP290 gene, located on chromosome 12q21.32, spans approximately 93.7 kb and contains 54 exons encoding a centrosomal and ciliary protein essential for photoreceptor function [11, 12]. Owing to its large genomic size and complex splicing architecture, molecular diagnosis of CEP290-associated disease remains challenging, particularly for variants affecting non-canonical splice sites, deep intronic regions, and structural rearrangements [10, 11]. Although CEP290 has been extensively investigated in inherited retinal degeneration, the vast majority of reported pathogenic variants are single-nucleotide variants, small insertions/deletions, or splice-altering variants. In contrast, intragenic structural variants are comparatively uncommon and have been described in relatively few studies [4]. Moreover, only a limited number of these structural variants have been resolved at nucleotide-level breakpoints, thereby limiting our understanding of the molecular mechanisms underlying their formation and pathogenic consequences [10, 13].
Here, we report a 4-year-old boy with severe early-onset retinal degeneration carrying compound heterozygous CEP290 variants, including a known pathogenic splice-site mutation (c.6012–2 A > G, maternal) and a novel large tandem duplication involving exons 31–53 (chr12:88050034–88089159 dup, paternal). Using trio-WGS, gap-PCR, and Sanger sequencing, we precisely mapped the duplication breakpoint and investigated the variant’s predicted functional impact and mutational mechanism. This study expands the structural variant spectrum of CEP290 and illustrats the value of breakpoint-level characterization in genetically unsolved IRD cases.
Materials and methods
Pedigree and participants
A 4-year-old male with early-onset retinal degeneration and his unaffected parents were recruited. Clinical evaluation included medical history, family history, visual acuity, visual fields, fundus photography, and optical coherence tomography (OCT). Ethical approval was obtained from the Institutional Review Board of West China Hospital, Sichuan University (approval No. 2021 − 190), and informed consent was obtained from the participant’s legal guardians.
Prior commercial ophthalmic gene panel testing
Prior to trio whole-genome sequencing (WGS), the proband underwent commercial targeted ophthalmic gene panel testing (OT050: Ophthalmic Disease Gene Panel-V5, GenCap capture probe V5.0, Nanjing Medical University Eye Hospital). This panel covered 1173 inherited retinal disease-related genes across a total 4 Mb target capture region, including CEP290 and other major ciliopathy and retinopathy disease genes. Library construction and high-throughput targeted capture sequencing were performed following standard manufacturer protocols.
The panel sequencing only identified a single heterozygous pathogenic splice-site variant c.6012–2 A > G in CEP290, while no additional pathogenic or likely pathogenic variants were detected in any other IRD-associated genes. Since autosomal recessive early-onset retinal degeneration requires biallelic pathogenic variants to establish a definitive molecular diagnosis, the presence of only one identifiable disease allele left the patient’s genetic diagnosis unresolved. Notably, targeted panel sequencing cannot reliably detect large intragenic copy number variants (CNVs) or resolve rearrangement breakpoints, as its capture design primarily focuses on exon-level single-nucleotide variants and small indels, with limited sensitivity for complex structural rearrangements. To capture the missing second pathogenic allele and fully characterize potential large structural variants undetectable by panel testing, we proceeded with trio WGS for comprehensive genome-wide genetic analysis.
Whole-genome sequencing and data analysis
Genomic DNA was isolated from peripheral blood samples of the proband and unaffected parents using an automated magnetic bead extraction workflow on the KingFisher Flex 96-well magnetic particle processor (Thermo Fisher Scientific, USA) with the MagPure Tissue/Blood DNA LQ kit (Magen, China). DNA concentration was quantified via the ExKubit dsDNA BR Assay Kit (Excell Bio, China), while purity and intactness were evaluated using a NanoDrop spectrophotometer and agarose gel electrophoresis, respectively. Qualified DNA was fragmented, subjected to end repair and adapter ligation for library construction. Paired-end 150 bp whole-genome sequencing was performed on the DNBSEQ-T7 platform (MGI, Shenzhen, China). The Burrows-Wheeler Aligner(BWA, v0.7.15) was used to map the reads to the human reference genome (GRCh38) [14].
An in-house variant-calling workflow, based on the GATK best practice, was developed for variant identification. Variants were annotated with information on variant quality, details, gene information, sequence database, allele state, and allele frequency [15]. For variant analysis, we applied the following filters: (1) Genotype quality ≥ 20, read depth ≥ 6, and alternate allele frequency > 0.1; (2) Popmax minor-allele frequency (MAF) ≤ 0.5% in the Genome Aggregation Database (gnomAD) (https://gnomad.broadinstitute.org) and retention of coding variants, splice regions, or selected pathogenic or likely pathogenic variants in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar) and Human Gene Mutation Database (HGMD) (https://www.hgmd.cf.ac.uk). The pathogenic potential of the identified variants was classified and interpreted according to the ACMG/AMP variant interpretation guidelines for genetic disorders [16]. SpliceAI(https://spliceailookup.broadinstitute.org) was applied to predict splicing alterations induced by intronic and splice-site variants, with a score ≧ 0.5 defined as evidence for high-impact splicing disruption. AutoPVS1(https://autopvs1.bgi.com) was applied to automate ACMG PVS1 criterion evaluation for loss-of-function variants at canonical GT-AG splice junctions, following the official ACMG PVS1 decision flowchart.
Additionally, we used CNVnator(v0.4.1) [17] and Manta(Illumina, v1.6.0) [18] to detect copy number variants (CNVs) in the WGS data. CNVs were filtered based on the following criteria: (1) Allele frequencies < 1% in gnomAD and the 1000 Genomes Project (https://www.1000genomes.org); (2) ≤ 50% reciprocal overlap with the Database of Genomic Variants (DGV) (http://dgv.tcag.ca); )(3) ≤ 25% overlap with repetitive and low-complexity regions. The pathogenicity of CNVs was interpreted in accordance with the ACMG/AMP variant interpretation guidelines [19].
Sanger sequencing and duplication breakpoint mapping
The variants identified in the proband through WGS were validated in affected proband and his unaffected parents using Sanger sequencing. To clarify, the family members included in this study were the proband’s father and mother, as they were the only available family members for genetic analysis at the time of sample collection. Primer sequences for polymerase chain reaction (PCR) amplification were designed using Primer3 software (http://primer3.ut.ee/), and the primer details can be found in Supplementary Table 1. The PCR products were analyzed by agarose gel electrophoresis to confirm quality and size. Subsequently, the PCR products were purified and subjected to Sanger sequencing using standard protocols.
Candidate structural variants were manually reviewed in Integrative Genomics Viewer (IGV, v2.17.4, Broad Institute). To validate the candidate CEP290 duplication detected by WGS, and determine the precise breakpoint, we designed gap-PCR primers spanning the predicted junction (forward primer at chr12:88050534; reverse primer at chr12:88089159) (primers listed in Supplementary Table 1). PCR amplification using genomic DNA of the proband and both parents was carried out in a 20 µL reaction mixture containing PrimeSTAR Max DNA Polymerase (Cat. No. R045Q, Takara Bio, Japan) under the manufacturer’s recommended conditions. PCR products were examined by agarose gel electrophoresis to confirm amplification quality, followed by purification and bidirectional Sanger sequencing on an ABI 3730X DNA Analyzer. The resulting chromatograms were analyzed to identify and define the exact duplication breakpoint region within the CEP290 gene.
Breakpoint analysis
Breakpoint coordinates were determined based on WGS read-pair and split-read signatures and refined by gap-PCR and Sanger sequencing. Genomic sequences flanking the upstream and downstream breakpoints were extracted from the GRCh38/hg38 reference genome.
Upstream and downstream breakpoint-flanking sequences were aligned using EMBOSS Needle (https://www.bioinformatics.nl/cgi-bin/emboss/needle, Recommended parameter settings: global alignment, EDNAFULL matrix, gap-open 10, gap-extend 0.5) to identify microhomology at the junction. Following previous studies of structural variant breakpoint analysis, microhomology was considered present when a contiguous identical sequence of at least 4 bp was identified within 10 bp of the breakpoint junction [20, 21].
GC content of the duplicated region (chr12:88,050,034–88,089,159) was calculated using the “GC Content” tool in the UCSC Genome Browser (http://genome.ucsc.edu/). RepeatMasker(http://www.repeatmasker.org/) was used to assess the density and composition of interspersed repetitive elements across the duplicated interval.
Results
Clinical findings
The proband presented with nyctalopia and progressive vision loss at 4 years old. Ophthalmic examination revealed retinal abnormalities consistent with presumed rod-cone dystrophy, including loss of the ellipsoid zone, centripetal RPE atrophy, mid-peripheral pigmentation, and abnormal autofluorescence. Systemic examination was normal. No other family members presented with similar ocular manifestations. Detailed clinical features and ophthalmic imaging findings are shown in Fig. 1.
Fig. 1.
Retinal imaging findings of the proband with CEP290-associated early-onset retinal degeneration.Optical coherence tomography (OCT) images of the left and right eyes demonstrate attenuation and loss of the ellipsoid zone, consistent with photoreceptor degeneration; Fundus autofluorescence (FAF) images show abnormal autofluorescence and centripetal retinal pigment epithelium (RPE) atrophy involving the posterior pole. BCVA, best-corrected visual acuity; LE, left eye; RE, right eye
Molecular findings
Trio-WGS identified a splice-site variant, NM_025114.4:c.6012–2 A > G, and a large duplication encompassing exons 31–53 of the CEP290 gene (chr12:88050034-88089159dup, GRCh38) (Fig. 2). The splice-site variant was maternally inherited, whereas the duplication was paternally inherited, consistent with a compound heterozygous genotype (Fig. 2).
Fig. 2.
Molecular characterization of the compound heterozygous CEP290 variants and breakpoint-resolved tandem duplication. (A) Trio whole-genome sequencing identified a 39,126-bp tandem duplication involving CEP290 exons 31–53. Gap-PCR and Sanger sequencing confirmed the head-to-tail orientation of the duplication and resolved the breakpoint junction at nucleotide resolution. The junction fused the 3′ end of intron 53 (chr12:88050034) with the 5′ end of exon 31 (chr12:88089159). Breakpoint sequence analysis demonstrated a 7-bp shared microhomology (AAATTCT) retained at the junction, a feature compatible with microhomology-mediated structural rearrangement.(B) Validation and segregation analysis of the canonical splice-site variant c.6012-2A>G by Sanger sequencing. The variant was detected in the proband and mother but was absent in the father, confirming maternal inheritance
The maternally inherited splice acceptor variant NM_025114.4:c.6012–2 A > G is a well-established pathogenic CEP290 variant. SpliceAI predicted complete disruption of the canonical splice acceptor site (score = 0.99), and AutoPVS1 supported a Very Strong (PVS1) loss-of-function classification. This variant is extremely rare, with a maximum population allele frequency of 4.56 × 10−⁵ in gnomAD v4.1. It is listed in ClinVar as a pathogenic variant and has been reported in two patients with CEP290-related disorders in compound heterozygotes with pathogenic variants [13, 22]. According to the ACMG/AMP guidelines [16], this variant is classified as pathogenic, based on evidence codes PVS1, PM2_Supporting, and PM3_Strong.
Gap-PCR and Sanger sequencing confirmed that the duplicated fragment is inserted within intron 53 of the CEP290 gene, spanning a total genomic length of 39,126 bp. The upstream breakpoint originated from the second nucleotide of amino acid 1301 in exon 31 and extended downstream into intron 53. This rearrangement is predicted to disrupt the normal reading frame, which would trigger premature termination and subsequent predicted nonsense-mediated mRNA decay (NMD). We performed searches against gnomAD v4.1, DECIPHER(v11.39, https://www.deciphergenomics.org) and DGV gold standard release 2020-02-25 on June 2026; no matching tandem duplication matching this CEP290 rearrangement was identified, and this variant has not been previously described in published literature. Notably, multiple known pathogenic variants have been documented in exon 54 of CEP290 in the ClinVar database, further supporting the pathogenicity of this duplication. Based on the ACMG/AMP framework and the ClinGen SVI recommendations for interpretation of intragenic tandem duplications [19, 23], the CEP290 exons 31–53 duplication was classified as likely pathogenic. Breakpoint analysis confirmed a tandem duplication, and the predicted transcript consequence is a frameshift leading to a premature termination codon, supporting application of PVS1_Strong. Together with PM2_Supporting and PM3, the variant was classified as likely pathogenic.
Breakpoint sequence analysis
Sequence-level analysis revealed a shared 7-bp microhomology sequence (“AAATTCT”) at the junction (Fig. 2A). Genomic context analysis of the duplicated segment (chr12:88050034–88089159, GRCh38) showed a low GC content of 33.99%, consistent with an A/T-rich region prone to replication fork instability. RepeatMasker analysis further indicated that 32.75% of the sequence was composed of interspersed repetitive elements, including: short interspersed nuclear elements (SINEs, 11.74%, predominantly Alu and MIR); long interspersed nuclear elements (LINEs, 12.59%, including LINE1 and LINE2); long terminal repeat retrotransposons (LTRs, 3.24%); and DNA transposons (5.04%, such as hAT-Charlie) (Supplementary Table 2).
Taken together, the presence of a short microhomology sequence at the breakpoint, the low GC content, and the high density of repetitive elements support a genomic environment conducive to non-recurrent structural rearrangements. These features are consistent with replication-based mechanisms such as FoSTeS/MMBIR, which have been associated with rearrangements characterized by microhomology at breakpoint junctions and non-recurrent breakpoint architecture. However, these findings are not specific to replication-based mechanisms, and alternative microhomology-mediated DNA repair pathways cannot be excluded.
Discussion
In this study, we identified a novel large tandem duplication encompassing CEP290 exons 31–53 with breakpoint validation by using trio-WGS and gap-PCR. This duplication, together with a maternally inherited splice-site variant (c.6012–2 A > G), forms a compound heterozygous genotype in the proband with early-onset severe retinal dystrophy characterized by nyctalopia, progressive visual field constriction, and macular atrophy. These findings expand the spectrum of structural variants in CEP290 and illustrate the diagnostic value in genetically unresolved recessive IRD cases.
Because the identified tandem duplication is predicted to disrupt the coding sequence downstream of exon 53 and is predicted to cause functional loss of exon 54, we further evaluated the pathogenic significance of exon 54 in CEP290-associated retinal degeneration. To assess the pathogenic relevance of exon 54 loss, we queried the ClinVar database and identified multiple curated pathogenic loss-of-function (LoF) variants-including nonsense, frameshift, and canonical splice-site mutations-within this exon. While these variants are associated with non-syndromic isolated retinal conditions including early-onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) consistent with our proband’s clinical presentation, they also exhibit broad phenotypic heterogeneity, with many carriers developing multi-system syndromes such as Bardet-Biedl, Joubert, and Meckel-Gruber syndromes. Collectively, these observations support the clinical relevance of loss-of-function variants involving exon 54 and suggest that disruption of this terminal coding region may contribute to CEP290-associated retinal degeneration (Table 1).
Table 1.
Summary of the reported CEP290 mutations in exon 54 and associated phenotypes to data
| Nucleotide change | Protein change | Non-syndromic phenotype (retina-only involvement) |
Syndromic phenotype (multi-system involvement) |
Clinvar ID |
|---|---|---|---|---|
| c.7209 + 2T > G | - | Leber congenital amaurosis | Joubert syndrome | 3,756,390 |
| Meckel-Gruber syndrome | ||||
| c.7209 + 1G > A | - | - | Bardet-Biedl syndrome 14 | 2,680,597 |
| c.7220_7223del | p.Lys2407fs | Retinal dystrophy | Bardet-Biedl syndrome 14 | 418,123 |
| Joubert syndrome | ||||
| Leber congenital amaurosis | Meckel syndrome, type 4 | |||
| Senior-Loken syndrome 6 | ||||
| c.7233del | p.Glu2412fs | - | Bardet-Biedl syndrome 14 | 1,419,020 |
| Joubert syndrome | ||||
| Meckel-Gruber syndrome | ||||
| Nephronophthisis | ||||
| c.7233dup | p.Glu2412fs | Leber congenital amaurosis | Bardet-Biedl syndrome 14 | 848,929 |
| Joubert syndrome 5 | ||||
| Meckel syndrome, type 4 | ||||
| Senior-Loken syndrome 6 | ||||
| c.7234del | p.Glu2412fs | - | Bardet-Biedl syndrome 14 | 1,324,055 |
| c.7240G > T | p.Glu2414Ter | - | Bardet-Biedl syndrome 14 | 3,241,103 |
| c.7248del | p.Phe2416fs | - | Bardet-Biedl syndrome 14 | 2,680,629 |
| c.7256 C > G | p.Ser2419Ter | - | Bardet-Biedl syndrome 14 | 2,680,608 |
| c.7256_7259del | p.Ser2419fs | - | Bardet-Biedl syndrome 14 | 2,680,581 |
| c.7263del | p.Phe2421fs | Leber congenital amaurosis | Bardet-Biedl syndrome 14 | 2,947,800 |
| Joubert syndrome 5 | ||||
| Meckel syndrome, type 4 | ||||
| Senior-Loken syndrome 6 | ||||
| c.7263dup | p.Glu2422Ter | Leber congenital amaurosis | Bardet-Biedl syndrome 14 | 1,387,697 |
| Joubert syndrome 5 | ||||
| Meckel syndrome, type 4 | ||||
| Senior-Loken syndrome 6 | ||||
| c.7264G > T | p.Glu2422Ter | Joubert syndrome | 1,448,988 | |
| - | Meckel-Gruber syndrome | |||
| Nephronophthisis | ||||
| c.7282_7283dup | p.Tyr2429fs | Leber congenital amaurosis | Bardet-Biedl syndrome 14 | 1,075,924 |
| Joubert syndrome | ||||
| Meckel syndrome, type 4 | ||||
| Nephronophthisis | ||||
| Senior-Loken syndrome 6 | ||||
| c.7287T > A | p.Tyr2429Ter | Bardet-Biedl syndrome 14 | 1,071,318 | |
| Joubert syndrome | ||||
| Leber congenital amaurosis | Meckel-Gruber syndrome | |||
| Nephronophthisis | ||||
| c.7282_7286dup | p.Tyr2429Ter | Joubert syndrome 5 | 1,698,978 | |
| - | Meckel-Gruber syndrome | |||
| Nephronophthisis | ||||
| c.7283_7286dup | p.Tyr2429Ter | Leber congenital amaurosis | Bardet-Biedl syndrome 14 | 956,518 |
| Joubert syndrome 5 | ||||
| Meckel-Gruber syndrome | ||||
| Nephronophthisis | ||||
| c.7288_7289dup | p.Asn2430fs | Leber congenital amaurosis | Bardet-Biedl syndrome 14 | 1,452,299 |
| Meckel-Gruber syndrome | ||||
| Nephronophthisis | ||||
| c.7289_7292dup | p.Tyr2431Ter | Joubert syndrome 5 | 1,076,282 | |
| - | Meckel-Gruber syndrome | |||
| Nephronophthisis | ||||
| c.7311del | p.Asn2438fs | - | Bardet-Biedl syndrome 14 | 2,680,574 |
| c.7318_7321dup | p.Leu2441fs | Retinal dystrophy | - | 866,473 |
| c.7324G > T | p.Glu2442Ter | Leber congenital amaurosis | Bardet-Biedl syndrome 14 | 1,071,910 |
| Joubert syndrome 5 | ||||
| Meckel syndrome, type 4 | ||||
| Senior-Loken syndrome 6 | ||||
| c.7327_7330delinsTATT | p.Glu2443_Lys2444delinsTyrTer | - | Bardet-Biedl syndrome 14 | 2,680,610 |
| c.7331del | p.Lys2444fs | Joubert syndrome 5 | 2,041,164 | |
| - | Meckel-Gruber syndrome | |||
| Nephronophthisis | ||||
| c.7328_7332dup | p.Val2445fs | Leber congenital amaurosis | Bardet-Biedl syndrome 14 | 663,263 |
| Meckel-Gruber syndrome | ||||
| Nephronophthisis | ||||
| Senior-Loken syndrome 6 | ||||
| c.7328_7332del | p.Glu2443fs | Leber congenital amaurosis | Bardet-Biedl syndrome 14 | 856,893 |
| Joubert syndrome 5 | ||||
| Meckel syndrome, type 4 | ||||
| Nephronophthisis | ||||
| Senior-Loken syndrome 6 | ||||
| c.7334dup | p.Leu2448fs | - | Bardet-Biedl syndrome 14 | 2,680,609 |
| c.7341dup | p.Leu2448fs(comp) | Leber congenital amaurosis | Bardet-Biedl syndrome 14 | 99,864 |
| Joubert syndrome 5 | ||||
| Meckel-Gruber syndrome | ||||
| Nephronophthisis | ||||
| Senior-Loken syndrome 6 | ||||
| c.7341del | p.Lys2447fs | Bardet-Biedl syndrome 14 | 1,076,473 | |
| Joubert syndrome 5 | ||||
| Leber congenital amaurosis | Meckel syndrome, type 4 | |||
| Nephronophthisis | ||||
| Senior-Loken syndrome 6 | ||||
| c.7341_7344dup | p.Ser2449fs | Rod-cone dystrophy | Joubert syndrome 5 | 830,333 |
| Leber congenital amaurosis | Meckel-Gruber syndrome | |||
| Nephronophthisis |
Notably, the splice-site variant c.6012–2 A > G has previously been reported in compound heterozygosity with other loss-of-function alleles, resulting in a spectrum of phenotypes including LCA and Joubert syndrome [13, 22]. In contrast, our patient presented with early-onset non-syndromic retinitis pigmentosa. This discrepancy suggests that, even for the same variant, differences in allelic context may substantially influence the resulting phenotype, highlighting the complex genotype-phenotype correlations in CEP290-associated disease [24, 25]. This early-onset severe phenotype also aligns with the established genotype-severity relationship in CEP290-associated disease: biallelic truncating variants typically cause early-onset RP or LCA, whereas missense variants are more commonly associated with milder cone-rod dystrophy (CORD) phenotypes [5, 24, 26].
Breakpoint sequence analysis revealed a 7-bp microhomology sequence (“AAATTCT”) at the junction of the duplication, within a region of low GC content and enriched repetitive elements. These characteristics are consistent with replication-based rearrangement mechanisms such as MMBIR and FoSTeS [21, 27, 28], which are known to generate non-recurrent structural variants with breakpoint microhomology. However, similar breakpoint signatures may also result from alternative microhomology-mediated repair pathways; therefore, the exact mechanism responsible for this duplication remains uncertain.
Beyond improving molecular diagnosis, precise characterization of pathogenic variants also has important therapeutic implications. Gene-based therapies for inherited retinal degeneration, particularly CEP290-associated disease, are rapidly advancing into clinical practice [29–31]. Accurate definition of the underlying mutational mechanism may therefore facilitate future precision therapeutic strategies.
Clinically, this study explains why the variant was missed by conventional genetic testing. Prior to WGS, the patient underwent a commercial ophthalmic gene panel test, which detected only the maternal splice-site variant (c.6012–2 A > G). Panel and exome-based methods are inherently limited in detecting complex structural variants, particularly those occurring within large, repeat-rich introns-a hallmark of CEP290. Our findings highlight the diagnostic value of WGS combined with targeted gap-PCR confirmation and provide a framework for evaluating genetically unresolved patients with suspected CEP290-related disease.
From a genomic architecture perspective, CEP290 is characterized by large intronic regions, relatively low GC content, and abundant repetitive elements. These features have been associated with genomic instability and may increase susceptibility to complex structural rearrangements arising through replication or repair-based mechanisms. Such genomic architecture may partly contribute to the diverse spectrum of pathogenic variants reported in CEP290-associated retinal disease [10, 21, 28].
Despite advances in next-generation sequencing, CNV detection and interpretation in CEP290 and other IRD-associated genes remain technically challenging. Short-read sequencing exhibits limited sensitivity for complex structural variants [3], and many identified CNVs lack functional validation, complicating variant classification under ACMG/AMP guidelines [19]. Emerging long-read sequencing and optical genome mapping technologies show great promise for resolving complex SVs and refining breakpoint resolution, but their widespread clinical implementation remains limited at present [32]. Therefore, orthogonal methods such as gap-PCR or ddPCR are essential complementary tools for confirming clinically relevant CNVs.
Despite the successful molecular diagnosis achieved in this study, several limitations should be acknowledged. First, full-field electroretinography was not performed. Therefore, the classification of the retinal phenotype as presumed rod-cone dystrophy was based on clinical presentation and multimodal retinal imaging findings rather than electrophysiological confirmation. Second, the predicted molecular consequences of the tandem duplication were inferred from genomic analyses and have not been experimentally validated at the RNA or protein level. Third, the mechanism of duplication formation remains uncertain. Although the breakpoint features are consistent with replication-based mechanisms such as MMBIR/FoSTeS, alternative microhomology-mediated repair pathways cannot be excluded. Finally, given the rarity of this tandem duplication, additional cases and larger cohort studies will be required to determine whether exon 31–53 duplication represents a recurrent rearrangement pattern in CEP290. Long-term clinical follow-up will further improve understanding of disease progression and genotype-phenotype correlations.
Taken together, through an integrated analysis spanning clinical genetics, structural variant biology, and genomic architecture, this study provides a detailed mechanistic and diagnostic characterization of a rare CEP290 tandem duplication. Precise characterization of structural variant breakpoints may improve molecular diagnosis, facilitate genotype-phenotype correlation, and provide valuable information for future gene-editing strategies targeting CEP290-associated retinal degeneration.
Conclusions
This study reports, for the first time, and precisely characterizes a novel large tandem duplication of exons 31–53 in the CEP290 gene. This variant, in compound heterozygosity with a maternally inherited pathogenic splice-site mutation (c.6012–2 A > G), is predicted to be the causal lesion underlying the patient’s early-onset severe retinal degeneration. We propose that the tandem duplication disrupts the CEP290 coding sequence and is predicted to result in loss of function through a truncating effect and/or nonsense-mediated mRNA decay. Our findings broaden the genotype-phenotype spectrum of CEP290-associated retinopathy and provide a molecular rationale for future targeted therapies against complex structural variants.
Supplementary Information
Acknowledgements
The authors express their gratitude to the patients, families, and scientists who made valuable contributions to this study. The research was supported by National Supercomputing Center in Chengdu.
Abbreviations
- IRD
Inherited Retinal Degeneration
- RP
Retinitis Pigmentosa
- LCA
Leber Congenital Amaurosis
- CORD
Cone/Cone-Rod Dystrophies
- WGS
Whole-Genome Sequencing
- OCT
Optical Coherence Tomography
- CNV
Copy Number Variation
- SV
Structural Variant
- ACMG/AMP
American College of Medical Genetics and Genomics/Association for Molecular Pathology
- MMBIR
Microhomology-Mediated Break-Induced Replication
- FoSTeS
Fork Stalling and Template Switching
Authors’ contributions
S.X.and J.G. contributed equally to this work; S.X., J.G., and Y.L. conceived and designed the study; W.X. was responsible for the follow-up of case data; S.X., J.G., M.L.,C.W., and X.W. performed experiments, including genetic sequencing, variant analysis, and functional assays; S.X., J.G., T.T., and Q.Z. analysed the data and performed statistical analyses; S.X. and J.G. drafted the manuscript; Y.L. and J.G. critically revised the manuscript for important intellectual content and supervised the study. All authors reviewed and approved the final version of the manuscript.
Funding
This work was supported by the 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (ZYJC20002).
Data availability
Data generated in this study are available within the manuscript and supplementary information files. The two pathogenic variants identified herein have been deposited in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) under accession numbers SCV007592818 and SCV007592819.
Declarations
Ethics approval and consent to participate
This study was performed in line with the principles of the Declaration of Helsinki. Ethical approval for this study was granted by the Institutional Review Board of West China Hospital of Sichuan University (2021 − 190). Informed consent was obtained from all participants or their legal guardians.
Consent for publication
Written informed consent for publication of their personal or clinical details along with any identifying images has been obtained from all the participants and the parents/legal guardians of minors.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Shumei Xu and Jia Geng contributed equally to this work.
Contributor Information
Huijun Yuan, Email: yuanhj301@163.com.
Yu Lu, Email: samuelluyu@163.com.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Data generated in this study are available within the manuscript and supplementary information files. The two pathogenic variants identified herein have been deposited in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) under accession numbers SCV007592818 and SCV007592819.