Dr. Poornachandra B, P14831, Dr. Shetty Bhujang K, Dr. Rohit Shetty, Dr. Naresh Kumar Yadav

Retinal dystrophies (RDs) are progressive and the major cause of untreatable blindness in both eyes, which is characterized by retinal degeneration including loss of retinal pigment epithelial cells and photoreceptors [1].  Studies have shown that [2] highest level of heterogeneity in Retinal Dystrophies, approximately 250 genes and their mutations were associated with various forms of retinal dystrophies. It has clinical manifestations ranging from mild (night blindness) to severe even with early onset (Leber congenital amaurosis) of retinal degeneration.  The most common form of inherited retinal dystophies is Retinitis pigmentosa (RP) with the prevalence of 1 in 3500–4000 individual. It can be inherited through a dominant mode (adRP) or autosomal recessive (arRP), or an X-linked mode of inheritance (XlRP)[3,4]. It affects mainly the photoreceptor cells in the retina, rapidly progressive at younger age of onset in arRP form, but adRP patients showed a late onset associated with clinically less severe form[3,4]. Cone rod dystrophies (CRDs) affect the impairment of vision at early adult life in 1/ 40,000 people. Clinical symptoms include color vision problem, decreased central vision due to loss of cone function followed by night blindness and defect in the peripheral visual fields because of rod dysfunction[5].Leber congenital amaurosis (LCA) (OMIM #204000) is a prenatal or early-onset childhood inherited retinal dystrophy associated with poor vision, and severe retinal dysfunction including rods and cone cells, that detects light in the retina.Another most frequent childhood or adolescence macular dystrophy is stargardt disease (STGD) had a frequency of 1 in 50,000. ABCA4 gene mutations were found to be most frequent in autosomal recessive forms. Congenital achromatopsia is an isolated cone dystrophy, which is inherited in an autosomal-recessive mode and causes complete loss of cone function, whereas rod functions were maintained properly throughout the disease course.

Retinal dystrophies often misdiagnosed due to genetic complexity and overlapping clinical phenotypes. To develop new therapeutic approaches and accurate genetic counselling of affected patients and their family members, it is important to know the detailed clinical diagnosis and genotype-phenotype correlations.

Mutation events that occur in gene-coding or controlregions can give rise to indistinguishable clinical presentations, leaving the diagnosing clinician with many possiblecauses for a given condition or disease. With next generation sequencing (NGS), clinicians are provided a fast, affordable, and thorough way todetermine the genetic cause of a disease. Although high throughout sequencing of the entire human genome is possible, researchers and clinicians are typically interested inonly the protein-coding regions of the genome, referred to asthe exome. The exome comprises just over 1% of the genomeand is therefore much more cost-effective to sequence thanthe entire genome, while providing sequence information forprotein-coding regions.

We performed targeted next-generation sequencing (NGS) in clinically confirmed 21 unrelated patients with different forms of retinal dystrophies and their selected family members using panel comprising 184 genes, which covered previously associated genes with retinal disease.

The prospective study was approved by the Institutional Review Board and was performed as per institutional ethics guidelines and in accordance with the tenets of the Declaration of Helsinki. Subjects were recruited for the study after obtaining informed written consent either from the patient or the guardian and family members. A total of 21 patients with hereditary retinal dystrophies from unrelated families from India were investigated. Age at the time presentation ranged between 5 – 31 years.

Detailed medical history was obtained, followed by clinical examination including best-corrected Snellen visual acuity (BCVA), slit-lamp examination, Gonioscopy, indirect ophthalmoscopy and fundus photography. Fundus autofluorescence (FAF) imaging with a confocal scanning laser ophthalmoscope (Spectralis, Heidelberg Engineering, Heidelberg, Germany) in all patients and selected family members was performed. Spectral domain optical coherence tomography (SD OCT; Spectralis, Heidelberg Engineering, Heidelberg, Germany) was also performed simultaneously in most of these patients and in pediatric cases a handheld SD-OCT (Envisu 2300, Bioptigen, DNC, USA) was performed. Electrophysiologic examinations were conducted according to the standards given by the International Society of Clinical Electrophysiology in Vision. 18, 19  Viking 5.0 Ganzfeld dome (Nicolet Biomedical Instruments, Madison, Wisconsin, USA) with a light-emitting diode for light stimulation was used for both electro-oculography and full-field electroretinography in selected patients.

The next generation sequencing was carried using a gene panel comprising 184 retinal specific genes. Sequencing results were analyzed by read mapping and variant calling in genes of interest, followed by their verification and interpretation.

The sequencing analysis revealed a total of 21 different mutations in patients with retinal dytrophies including Leber’s congenital amaurosis , cone-rod dystrophy, Retinitis pigmentosa,Achromatopsia and Stargardt’s disease. Among these seven mutations were unreported and fourteen variants were previously associated with Retinal Dystrophies.These nucleotide changes were not present in 100 normal controls analyzed. We add seven novel mutations with existing spectrum of gene mutations identified in Indian patients with the characteristic features of Retinal Dystrophies, which provide further information on the genotype/phenotypic correlation

Genetic analysis of Retinal Dystrophypatients  by using  targeted next generationsequencing analysis would be an efficient method with low cost, might be useful for accurate clinical diagnosis, genetic counselling and to guide familial or isolated patients with Retinal Dystrophies.

References

1. den Hollander, A.I.; Black, A.; Bennett, J.; Cremers, F.P. Lighting a candle in the dark: Advances in genetics and gene therapy of recessive retinal dystrophies. The Journal of clinical investigation 2010, 120, 3042-3053.
2. Retinal information network. Accessed September 2017. Available on : https://sph.uth.edu/retnet/
3. Berger, W.; Kloeckener-Gruissem, B.; Neidhardt, J. The molecular basis of human retinal and vitreoretinal diseases. Progress in retinal and eye research 2010, 29, 335-375.
4. Hartong, D.T.; Berson, E.L.; Dryja, T.P. Retinitis pigmentosa. Lancet 2006, 368, 1795-1809.
5. Hamel, C.P. Cone rod dystrophies. Orphanet journal of rare diseases 2007, 2, 7.