The existence of high clinical heterogeneity ensuing from intricate genetics has been demonstrated for LCA etiology. Hence, CES was performed for ten LCA patients with variable phenotypes to unravel causative mutations contributing to disease pathogenesis.
RPE65 is one of the most common LCA candidate genes and mutation in this gene contributes to 3–16% of LCA cases worldwide . Patient AS01 was identified with a pathogenic frameshift mutation in the carotenoid oxygenase domain of the RPE65 gene, which creates a stop codon at exon 5, and thus produces a truncated protein of 129 amino acids residues. It might have partial function compared to the wild-type protein of 533 amino acids or may undergo nonsense-mediated mRNA decay. This category of gene mutations causes a deficiency of 11-cis-retinal especially in rods compared to cones, leading to nyctalopia [3, 8]. Patients with RPE65 mutation also exhibit some extent of improvement in visual acuity over the first decade of life, but it will eventually deteriorate in the later decades . Our patient also had a history of nyctalopia and showed transient improvement in visual acuity, consistent with earlier reports. The same mutation was formerly identified in South Indian LCA patients among many other mutations identified at this c.361 codon position, which makes it a hot spot for Indian LCA patients [5, 7, 15].
LCA5 consists of 697 amino acids, which encodes highly conserved ciliary protein Lebercilin. Despite its wide expression in human tissues, LCA5 mutations are restricted to only cause retinal dysfunction with a prevalence rate of 1–2% . The majority of mutations reported with the LCA5 phenotype are null mutations . Our study identified different homozygous null mutations in two unrelated patients AS02 [17, 18] and AS03. Patient AS02 below 10 years of age showed mild improvement in vision, while patient AS03 was legally blind at 11 years of age. It was reported that patients with LCA5 mutations showed improvement in vision and eventually decline after the first decade of life . Even though Patient AS02 showed normal macula in fundoscopy and preserved central macular outer retinal layer in optic coherence tomography, increased autofluorescence at the macula was observed, suggesting increased metabolic activity of the retinal pigment epithelium. A marbled fundus was observed in patient AS03, as seen in the CEP290-related LCA phenotype. Patient AS03 developed a posterior subcapsular cataract, reported as a common feature of patients with LCA5 mutations .
CRX (cone-rod homeobox) is often reported to cause LCA in an autosomal dominant pattern . The present study also provides evidence by identifying a novel heterozygous frameshift mutation in patient AS04. Segregation analysis revealed the same mutation in the affected mother, while absent in normally sighted father and younger sibling, indicating that the patient inherited a disease-related mutation from his mother. CRX consists of 299 amino acid residues. Whereas in the proband AS04, frameshift shift mutation in exon 4 removes the native stop codon, as a result of which a larger open reading frame consisting of 369 amino acids is produced. Earlier reports on CRX mutations in the index cases have shown thinned, abnormal lamellar structure, and macular atrophy without noticeable signal of inner and outer segments junction in SD-OCT . Similarly, patient AS04 had a lack of lamination and complete loss of the outer retinal layer.
Individuals affected by PRPH2 mutation are known to have pattern dystrophy (butterfly-shaped pigment dystrophy and Adult-onset foveomacular vitelliform dystrophy) with a broad spectrum of clinical appearance, LCA and retinitis pigmentosa [22, 23]. Patient AS05 carried a reported missense mutation in the cytoplasmic domain of PRPH2 . HOPE predicted that the wild-type residue proline is very rigid and required special conformation of the protein backbone. Thus, the mutation at that position might affect protein function by disrupting special conformation . The vitelliform lesion is the most commonly encountered clinical presentation of adult-onset foveomacular vitelliform dystrophy and LCA, due to PRPH2-mediated phenotypes [22, 23]. In this study, patient AS05 also demonstrated early retinal defects with vitelliform lesions, consistent with the abovementioned studies.
Interestingly, CES analysis of patient AS06 revealed a novel homozygous missense mutation in the CEP290 gene. The HOPE tool has predicted that the mutation is present in the conserved region required for interaction with IQCB1 (IQ Motif Containing B1). This mutation introduces more hydrophobic residues, which may affect the hydrogen-bond formation and results in loss of interactions with other molecules . Mutation in CEP290 leads to LCA, Bardet–Biedl syndrome, Senior–Loken syndrome and Joubert syndrome . Unlike the Western population, where CEP290 mutations are the most common cause for LCA with an estimated prevalence of 15–20% [4, 25], to date no CEP290 mutations have been reported from the Indian population [13, 26]. At the age of 11 years, patient AS06 developed a cataract and presented with marbled fundus as reported in earlier studies [27, 28]. However, the presence of systemic anomalies like head nodding and secondary behavioral changes in the patient indicates an association with the abovementioned syndromes, therefore AS06 was advised to undergo complete systemic evaluation including an MRI scan and ultrasound to better understand the disease pathogenesis in this patient. However, due to kidney failure, the patient was deceased. The patient’s younger sibling (2 years) also presented with similar ocular features, head nodding and secondary behavioral changes. Since the sibling was identified with the same mutation, being strictly monitored, we hope to identify related syndromes and offer treatment early.
Mutation in ALMS1 was reported to cause Alstrom syndrome and LCA [29, 30]. One of our patients AS07 carried a homozygous frameshift mutation in ALMS1. Indeed, so far ALMS1 mutation-specific phenotypes have not been described extensively for LCA patients. However, Xu and colleagues have reported homozygous ALMS1 null mutation in six LCA cases with early-onset retinal degeneration, visual acuity from light perception to no light, high hyperopia, roving eye movement, oculo-digital sign, undetectable ERG and tapetal fundus . In agreement with this study, our patient also developed these clinical features, but the fundus revealed a pink disc, thread-like arteriolar attenuation, greyish desaturated appearance and wrinkling of the inner limiting membrane at the macula. The patient did not demonstrate any typical signs of Alstrom syndrome until now, and thus was advised to return for regular clinical assessments for any systemic abnormalities.
IFT80 is a component of Intra Flagellar Transport complex B, which is essential for the assembly and maintenance of motile and sensory cilia . IFT80 mutants underlie Jeune syndrome, an autosomal recessive disease characterized by the constricted thoracic cage, respiratory insufficiency, cystic renal disease, polydactyl disease and retinal degeneration. Patients with Jeune syndrome are likely to develop retinal dystrophies within a few months of birth [32, 33]. In the present work, one of the twins AS08 subjected to CES was identified with a missense mutation in the IFT80 gene. Later, the same mutation was found in another twin through segregation analysis. Both twins were presented with typical LCA clinical features as poor vision at an early stage, multiplanar nystagmus and non-recordable ERG. At 11 years, the patient observed with marbled fundus, similar to CEP290-associated LCA patients. The patient has not yet developed any other symptoms of Jeune syndrome except for the LCA phenotype. Many ciliopathy genes, such as CEP290, IFT140, and IQCB1 initially reported to cause syndromic retinal degeneration, but were later identified as having vital roles in LCA pathogenesis as well [25, 34, 35]. Similarly, the IFT80 gene might also be contributing to the LCA phenotype.
“The patient AS09 was initially diagnosed with LCA as she presented with poor visual acuity, multiplanar nystagmus, oculodigital sign, and non-recordable ERG at the age of 1.75 years. Interestingly, molecular diagnosis identified a homozygous frameshift mutation in exon 4 of the RP1 gene, documented to cause retinitis pigmentosa. Previous studies have shown that the mutation in exon 4 of RP1 causes retinitis pigmentosa in early life, as it encodes 85% of the protein . Similarly, the null mutations in exon 4 of RP1 might cause retinitis pigmentosa in the early stages of life. Thus, based on the genetic finding, the patient was re-defined with clinical diagnosis of autosomal recessive severe early-onset retinitis pigmentosa.”
The patient AS10, who was clinically diagnosed with LCA, was not identified with any underlying variant. In addition, the patient was also noted with dry, scaly skin and hyperpigmented knuckles. Whole exome or genome sequencing is required to understand the genetic cause of this disease phenotype.