Dr. POORNACHANDRA B.

Dr. ROHIT SHETTY, Dr.Naresh Kumar Yadav, Dr.Shetty Bhujang K

Semi Finals

Abstract

AIM: To explore changing paradigms in management of Retinal Dystrophies

METHODS: All patients underwent comprehensive evaluation, imaging and genetic testing to identify causative mutations. Blood samples were analysed for targeted gene sequencing using retinal disease panel. Functional analysis of novel mutations and therapeutic potentials tested in vitro. RESULTS:Out of 44 cases studied we had all spectrum of retinal dystrophies. All patient had relevant mutations, 9 were unreported novel mutations. Mutations had prognostic significance. Some had mutation for which clinical trial is underway. Effect of mutations on protein function was assessed in vitro. Sub-retinal delivery modality was developed in rodent models. CONCLUSION: Comprehensive analyses of retinal dystrophies helps patients to make better choices, understand the prognosis and to know what future holds for them. Understanding gene functions and in vivo methods of delivering therapeutics is essential for better treatments

Full Text 

Introduction

Retinal dystrophies (RD) are progressive and one of the major causes of untreatable blindness in both eyes. It is characterized by retinal degeneration including loss of retinal pigment epithelial cells and photoreceptors [1].  Studies [2] have shown that highest level of heterogeneity, approximately 250 genes and their mutations were associated with various forms of RD. Clinical manifestations range from mild (night blindness) to severe visual impairment, even with early onset (Leber congenital amaurosis) of retinal degeneration.  The most common form of inherited RD is Retinitis pigmentosa (RP) with a prevalence of 1 in 3500–4000 individuals. 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 show 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 colour 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 involving rods and cone cells, that detects light in the retina. Another frequent childhood or adolescence macular dystrophy is Stargardt disease (STGD) with 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.

RD are 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.

Materials and methods 

This was an observational study. Informed written consent either from the patient or the guardian and family members was documented. A total of 45 patients with hereditary retinal dystrophies from unrelated families from India were investigated. Age at the time presentation ranged between 5 – 31 years (median – 18) with Male : Female – 31 : 14. We performed targeted next-generation sequencing (NGS) in clinically confirmed 21 unrelated patients with different forms of RD and their selected family members using retinal dystrophy panel which covered previously associated genes with retinal disease. Sequencing results were analyzed by read mapping and variant calling in genes of interest, followed by their verification and interpretation.

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.

Sequencing results were analyzed by read mapping and variant calling in genes of interest, followed by their verification and interpretation.

Results and experimental analyses

The sequencing analysis revealed a total of 45 different mutations in patients with RD including Leber’s congenital amaurosis , cone-rod dystrophy, Retinitis pigmentosa, Achromatopsia and Stargardt’s disease. Among these nine mutations were unreported and fourteen variants were previously associated with RD. These nucleotide changes were not present in 100 normal controls analyzed. We add nine novel mutations with existing spectrum of gene mutations identified in Indian patients with the characteristic features of RD, which provide further information on the genotype/phenotypic correlation.

Detailed molecular and functional analysis of the genetic variants is critical to further our understanding of the disease process and clinical prognosis (6). Further, treatment of IRDs requires gene therapy approaches. Several studies are therefore underway using a variety of retinal dystrophy animal models to establish their efficacy (7). The gene delivery modality of choice is recombinant AAV vector, expressing the therapeutic genes. These are typically delivered by the sub-retinal route, followed by functional analysis between 6-12 weeks post transduction. AAV mediated delivery can transduce RPE as well as photoreceptors. (8)

A comprehensive approach is important to diagnose and label hereditary RD. This will help to guide patients regarding the prognosis, lifestyle modification and not to mis-label treatable conditions as dystrophies. Our initial mouse model based experiments will also help to guide familial or isolated patients with Retinal Dystrophies about future prospects of gene therapy.

References

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2. Retinal information network. Accessed September 2017. Available on : https://sph.uth.edu/retnet/

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