Identification and Segregation Analysis of LCA5 Variants in Two Pakistani Families with Leber Congenital Amaurosis

Leber Congenital Amaurosis (LCA) is a severe, early-onset retinal dystrophy characterized by profound visual impairment from birth along with nystagmus and abnormal electroretinogram (ERG) responses (Munir et al., 2024). LCA primarily affects photoreceptors, has a prevalence ranging from 1 in 372 in certain rural South Indian populations to 1 in 9000 in Korea (Tehreem et al., 2022). Though this prevalence varies globally, LCA remains a significant cause of inherited childhood blindness worldwide (Sato et al., 2020). Approximately, 5% of all inherited retinal dystrophies (IRDs) are caused by LCA and is considered to be the most severe form (Skorczyk-Werner et al., 2023). The genetic etiology of LCA is highly heterogeneous, with mutations in over 28 genes reported till now to be associated with the LCA phenotype, showing the complex genetic landscape underlying this condition (Tareen et al., 2025). Among these genes, LCA5, encoding the centrosomal protein lebercilin, is associated with the severe form of LCA associated with its mutations (Skorczyk-Werner et al., 2023). Lebercilin, a ciliary protein, is widely expressed in ciliated epithelia throughout the body however, mutations in LCA5 typically result in a retinal dysfunction in humans (Faber et al., 2023). In the mouse retina, Lca5 mRNA expression increases during development within photoreceptor cells, indicating specialized early stage function of Lebercilin in these cells (Faber et al., 2023).

The LCA5, encoding the lebercilin protein is essential for outer segment maintenance and photoreceptor survival (Den Hollander et al., 2007, 2008). Mutations in LCA5 disrupt ciliary transport mechanisms hence, causing photoreceptor dysfunction and degeneration (Jacobson et al., 2009; Mackay et al., 2013). Although LCA5 mutations are a rare cause of LCA globally, their incidence and mutation spectrum in the Pakistani population remain understudied.

This study describes the identification and detailed segregation analysis of two LCA5 frameshift variants NM_001122769.3 c.1435dupC p.(Gln479Profs*16) and c.1151delC p.(Pro384Glnfs*18) segregating in two unrelated Pakistani families, LUVI-31 and LUPCG18 respectively. Using genetic, bioinformatic, and protein structural analyses, we provide evidence supporting the pathogenicity of these variants and their role in LCA related disease etiology.

This study was approved by the ethical committee of National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore. Two unrelated Pakistani families, LUPCG18 and LUVI-31, were recruited from the Sindh province of Pakistan. Both families presented with vision impairment (HP: 0001133), nystagmus (HP:0000639), and photophobia (HP:0000613). Comprehensive clinical evaluations were conducted including detailed ophthalmological examinations and systemic phenotypic assessments including skeletal, behavioral and neurological features. Blood samples were taken after the written informed consents were obtained from all participants in accordance with institutional ethical guidelines.

Genomic DNA was extracted from peripheral blood samples using the organic extraction method. Whole exome sequencing (WES) was subsequently performed, and variants were filtered based on parameters as outlined previously (Ghaffar et al., 2024). Prioritized candidate variants were then validated and segregated in family members through Sanger sequencing.

The UCSC Genome Browser (Perez et al., 2025) and MetaDome protein (Wiel et al., 2019) prediction tool were utilized to generate bar diagrams illustrating gene and protein features, respectively. MetaDome was further used to assess the mutation tolerance ratio at specific amino acid positions. PolyPhen-2 (Adzhubei et al., 2013) was used to evaluate the conservation scores of both variants. Transcriptomic expression levels in human retinal organoids were analyzed using the UCSC Cell Browser (Perez et al., 2025) to identify impacted cell types. Protein-protein interaction analyses, including STRING (Szklarczyk et al., 2023) and binary interaction networks (Orchard et al., 2014), were performed to identify LCA5 protein-associated interacting proteins involved in disease pathogenesis. For structural insights, 3D protein modeling was conducted by extracting Protein Data Bank (PDB) files from AlphaFold (Jumper et al.,2014), followed by analysis with PyMOL to assess effects on hydrogen bonding and overall protein conformation.

In family LUPCG18, a rare frameshift deletion (NM_001122769.3) c.1151delC, previously reported (Munir et al., 2024), was identified (Fig. 1B). This variant occurring in exon 8 of LCA5 leads to premature truncation of Lebercilin in non-structural domain region (Fig. 2A).

Similarly for family LUVI-31, a novel variant (NM_001122769.3) c.1435dupC was segregated with the disease in an autosomal recessive pattern (Fig. 1A). The variant occurs in exon9 resulting into a frameshift mutation causing a premature protein truncation in a non-structural domain of the protein (Fig. 2A). Hence, both variants were homozygous in affected individuals and heterozygous in unaffected carriers, supporting recessive inheritance (Fig. 1A).

MetaDome scores for the mutant amino acid variation point to be tolerant with the score of 1.18 for p.(Pro384Glnfs*18) and 1.39 for p.(Gln479Profs*16) (Fig. 2B). However, PolyP100 predicts both mutations to be damaging with extremely rare population allele frequency (Table I).

Protein modeling for p.(Pro384Glnfs*18) does not show any hydrogen bond impact however, as proline is a ringed structure it provides more stability and rigidity to protein site and produces sharp curves for protein folding whereas glutamine has a linear structure and is more flexible amino acid as compared to proline. Hence, this mutation occurs in the loop region of the protein followed by early truncation resulting in structural instability and loss of function (Fig. 3A, B). p.(Gln479Profs*16) on the other hand results in into loss of three hydrogen bonds when Gln479 changes to Pro479. Wild type Gln479 makes two bonds with Asp475, one with each Arg476 and Arg482, respectively, and two with Asn483. When mutation was created from Gln479 to Pro479 a single hydrogen bond of 3.0 Å with Arg476 and two bonds with the length of 3.3 Å and 3.4 Å with Asp475 was lost (Fig. 3 A, C). Also, as described above proline is a more rigid amino acid causing kinks in proteins which can impact protein folding and stability.

Expression analysis was done on dataset of 285,441 single cells from retinal organoids with fully function retinal layers and synapse activation among cells, available at UCSC cell browser. Data shows overall higher expression of LCA5 in rods, cones, amacrine, retinal pigmented epithelium (RPE) cells, astrocytes and Muller cells (Fig. 4A, B).

STRING analysis showed major proteins involved in retinal development and function to be interacting with Lebercilin such as RDH12, SPATA7, RD3, AIPL1, RPGRIP1 and USH2A (Fig. 4C). Binary interaction shows a number of proteins involved in LCA phenotype along with LCA5 (Fig. 4D).

This study expands the mutational landscape of LCA5 by providing evidence of two pathogenic frameshift variants NM_001122769.3 c.1151delC (p.Pro384Glnfs*18) and c.1435dupC (p.Gln479Profs*16), segregating in two unrelated Pakistani families LUPCG18 and LUVI-31 diagnosed with LCA. Both variants were found to be causative for the disease in an autosomal recessive pattern of inheritance as shown through pedigree analysis (Fig. 1).

Our findings align with previously reported mutations and their phenotype are consistent with previous reported individuals carrying mutations in LCA5 showing phenotype of vision impairment (HP:0001103), nystagmus (HP:0000639), and photophobia (HP:0000613) (Corton et al., 2014; Den Hollander et al., 2007; Li et al., 2009; Mackay et al., 2013; Zobor et al., 2023). The identification of a novel variant (c.1435dupC) highlights the need for further population-based screening to help identify more mutations in LCA5 causative for LCA.

Frameshift mutations, similar to those identified in our study, usually result in premature truncation of the Lebercilin protein and loss of normal function (Ramprasad et al., 2008). Our Protein modeling and in silico analyses predicted that LCA5 protein variants p.(Pro384Glnfs*18) and p.(Gln479Profs*16) disrupt protein stability and folding, either due to absence structurally functional

regions or by altering amino acids responsible for hydrogen bond formation like for variation p.(Gln479Profs*16) three essential hydrogen bonds were lost in protein helical structure, while proline substitutions leads to cause local rigidity and kinks within the mutant LCA5 protein structure.

Mutation tolerance scores from MetaDome show mutant amino acid regions to be tolerant to amino acid changes (Fig. 2) however, the conservation analyses with PolyPhen-2 supported the likely deleterious nature of these variants (Table I), predicting their evolutionary constraint to LCA5 loci and supporting their pathogenicity to cause disease.

These structural disruptions impair Lebercilin’s function at the photoreceptor connecting cilium, disrupting protein trafficking essential for outer segment maintenance and phototransduction (den Hollander et al., 2007). Loss of Lebercilin thus leads to photoreceptor degeneration characteristic of LCA5-related LCA. Accurate variant identification supports patient stratification for emerging gene augmentation therapies, such as AAV-delivered LCA5 replacement vectors in preclinical development, and antisense oligonucleotide approaches to mitigate nonsense-mediated decay (Faber et al., 2023; Russell et al., 2022; Collin et al., 2012).

Single-cell transcriptomic profiling of human retinal organoids shows significant high RNA expression of LCA5 across all major retinal cell types, including rods, cones, RPE, astrocytes, and Müller cells (Fig. 4A, B). These results align with the previous studies highlighting Lebercilin’s predominant role in photoreceptor maintenance and ciliary function (Li et al., 2025). Most people with Lebercilin mutations only show symptoms in the eyes, even though the protein is found throughout the body, this is likely because Lebercilin is especially critical for developing and maintaining ciliary function in retina (Den Hollander et al., 2007). 

Interaction network analyses using STRING revealed extensive protein-protein interaction clusters, with LCA5 associated with other essential retinal dystrophy proteins such as RDH12, RPGRIP1, AIPL1, and USH2A (Fig. 4C, D). Disruption of these networks due to mutations could affect retinal structure and function.

According to Leiden Open Variation Database (LOVD) 100 different variants are reported till date, predominantly frameshifts and nonsense mutations causing loss of function for LCA5. The identification of a LCA5 novel variant p.(Gln479Profs*16) identified in this study signifies the need and importance of screening such genes with time to identify new genetic mutations, especially in underrepresented populations.

However, this study lacks functional characterization of the identified variants, and future studies are required to prove their actual pathogenicity.

This study identified two pathogenic frameshift variants in LCA5 in Pakistani families affected by LCA. Both variants, c.1151delC and c.1435dupC (NM_001122769.3), co-segregate with autosomal recessive inheritance and cause early-onset vision impairment (HP:0001103), nystagmus (HP:0000639), and photophobia (HP:0000613). Both the variants were predicted to be causing functional loss of protein through in silico analysis. Overall, this study not only expands the known LCA5 mutational spectrum but also demonstrates the value of combined clinical, genetic, and in silico approaches for variant interpretation. Hence, these findings support future genotype-driven diagnostic and therapeutic strategies in IRDs.

We would like to thank family members of family LUPCG18 and LUVI-31 for participating in this study.

This study was not supported by any funding grant.

This study received approval from the Institutional Review Boards at the National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan and Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan.

This study was approved by the ethics committee of National Centre of Excellence in Molecular Biology, University of the Punjab Lahore (No: IRB-ID/2023), and the informed consent was obtained from guardian of the patients and all participants.

Informed consent was obtained from all the individuals involved in the study.

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

The authors have declared no conflict of interest.