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Sankara Nethralaya
Sankara Nethralaya
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Department of Genetics and Molecular Biology

Rhodopsin (RHO )gene screening:

Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited diseases that result in progressive retinal degeneration, characterized by visual field constriction and night blindness. Mutations in the rhodopsin gene are a major contributor to various retinopathies such as retinitis pigmentosa and X-linked congenital stationary night blindness. Rhodopsin, also known as visual purple, is a biological pigment of the retina that is responsible for both the formation of the photoreceptor cells and the first events in the perception of light. A total of 103 mutations in rhodopsin are linked to RP to date and the phenotypes range from severe to asymptomatic. And there are reports stating, Vitamin A supplementation may confer therapeutic benefit by stabilizing certain mutant opsins through increased availability of the chromophore. Here, we screen the patients with retinitis pigmentosa for mutations in RHO gene by a standardized PCR based direct sequencing.

Kumaramanickavel G, Maw MA, Denton MJ, John S, Srikumari CRS, Orth U, Ralph O, Gal A. Missense rhodopsin mutation in a family with recessive RP. Nature Genetics; 1994:8: 10-11.
Gandra M, Anandula V, Authiappam V, Sundaramurthy S, Raman R, Bhattacharya S, Kumaramanickavel G. Retinitis pigmentosa: mutation analysis of RHO, PRPF31, RP1, and IMPDH1 genes in patients from India. Mol Vis. 2008 Jun 14; 14:1105-13. PMID: 18552984 [PubMed – indexed for MEDLINE]

RPE65 gene screening:

Leber Congenital Amaurosis (LCA) and Retinitis Pigmentosa (RP) are inherited degenerative diseases of the retina. LCA is characterized by bilateral congenital blindness. RP is characterized by night blindness, with age of onset varying from childhood to middle age, and progressing to constriction of the peripheral visual field and, eventually, to loss of central vision. Mutations in the RPE65 gene have been associated with Leber's congenital amaurosis type 2 (LCA2) and retinitis pigmentosa. The retinal pigment epithelium-specific 65 kDa (RPE65) protein is located in the retinal pigment epithelium and is involved in the conversion of all-trans retinol to 11-cis retinal during photo-transduction, which is then used in visual pigment regeneration in photoreceptor cells. Studies in animals and detailed human studies of photoreceptor layer integrity have led to gene therapy trials in LCA caused by mutations in RPE65. Jacobson et al in 2012 reported the safety and efficacy of those trials in 15 Children and adults followed up to 3 years where visual function improved in all patients to different degrees and improvements were localized to treated areas. Hence we provide a diagnostic testing for RPE65 mutations in patients with retinitis pigmentosa and Lebers congenital amaurosis. The testing involves a PCR based direct sequencing of all 14 exons in RPE65 gene in patients.

Pre-PCR facility
Kumaramanickavel G, Maw MA, Denton MJ, John S, Srikumari CRS, Orth U, Ralph O, Gal A. Missense rhodopsin mutation in a family with recessive RP. Nature Genetics; 1994:8: 10-11.
Kar B, John S, Kumaramanickavel G. Retinitis pigmentosa in India: a genetic and segregation analysis. Clinical Genetics; 1995: 47: 75-79.
Gu SM, Thompson DA, Srikumari CR, Lorenz B, Finckh U, Nicoletti A, Murthy KR, Rathmann M, Kumaramanickavel G, Denton MJ, Gal A. Mutations in RPE65 cause autosomal recessive childhood-onset severe retinal dystrophy. Nature Genetics; 1997: 17:194 -197.
Finckh U, Xu S, Kumaramanickavel G, Schurmann M, Mukkadan JK, Fernandez T, John S, Weber JL, Denton MJ, Gal A. Homozygosity mapping of autosomal recessive retinitis pigmentosa locus (RP22) on chromosome 16p12.1-p12.3. Genomics; 1998 48:341-345.
Madhavan C, Bhende P, Lekha G. Vasanthi SB, Kumaramanickavel G. Retinitis Pigmentosa patients with Sickle Cell Disease and Dextrocardia and Situs Inversus Syndrome. Indian Journal of Ophthalmology; 2001:49:193-95.
Kumaramanickavel G, Selvaraj R, Rathanasabapathy V (1991). Retinitis pigmentosa: A genetic analysis. Afro-Asian J of Ophthalmology. Vol X: 20-25.
Joseph B, Anuradha S, Soumittra N, Vidhya A, Shetty NS, Uthra S, Kumaramanickavel G. RPE 65 gene: Multiplex PCR and Mutation screening in patients from India having retinal degenerative diseases. Journal of Genetics; 2002:81:1.

Cyp1b1 gene screening for glaucoma:

Primary congenital glaucoma (PCG) is a recessively inherited disorder of the eye with variable penetrance. The trabecular meshwork and anterior chamber angle of the eye are affected, leading to the obstruction of aqueous outflow, increased intraocular pressure (IOP) and optic nerve damage. The onset of this disease is seen in the neonatal or early infancy period and, if untreated, results in an irreversible blindness. Genetic heterogeneity is well documented in PCG. At the GLC3A locus that harbors the human cytochrome P450 gene CYP1B1, more than 40 different mutations that cause PCG have been identified, indicating a high degree of allelic heterogeneity. CYP1B1 is proposed to be the major gene for disease causing mutations in primary congenital glaucoma. Cytochrome P450 1B1 is an enzyme that metabolizes a signaling molecule involved in eye development, possibly a steroid. Screening of the patients for CYP1B1 mutations will be helpful in assessing the carrier status of the gene and prenatal diagnosis.

Ramprasad VL, George R, Sripriya S, Nirmaladevi J, Vijaya L, Kumaramanickavel G. Molecular genetic analysis of a consanguineous South Indian family with congenital glaucoma: Relevance of genetic testing and counseling. Ophthalmic Genetics 2007 Jan-Mar; 28(1):17-24.

MYOC gene screening for open angle glaucoma:

Primary open-angle glaucoma is one of the leading causes of blindness in the world. It is a neurodegenerative disorder characterized by progressive excavation of the optic discs due to loss of retinal ganglion cells. It is usually associated with elevation of intraocular pressure (IOP). Based upon the age of diagnosis, primary open-angle glaucoma can be sub-classified to either juvenile-onset primary open-angle glaucoma (JOAG) or adult-onset primary open-angle glaucoma. JOAG is a relatively rare form of primary open angle glaucoma that occurs in children and young adults. Strong evidence indicates that genetic factors play a role in the pathogenesis of glaucoma. To date, three genes, namely myocilin (MYOC), optineurin (OPTN), and WD repeat-containing protein 36 (WDR36), have been reportedly linked to POAG. MYOC was the first gene to be identified as responsible for POAG. Almost 80 mutations have been found in MYOC. MYOC encodes the protein myocilin, which is normally secreted into the aqueous humor of the eye to control the intraocular pressure through its cytoskeletal action in the muscle tissue of the ciliary body. MYOC mutations, which cause myocilin to accumulate in the cells of the trabecular meshwork increasing the intra ocular pressure resulting in glaucoma. This genetic testing provides pre-symptomatic molecular diagnosis for members of a family and is useful for further genetic counseling with the family.

PCR facility
Sripriya S, Uthra S, Sangeetha R, George RJ, Hemamalini A, Paul PG, Amali J, Vijaya L, Kumaramanickavel G. Low frequency of Myocilin mutations in Indian POAG patients. Clinical Genetics Apr 2004 (4) 333 – 337.

Pax6 gene screening for aniridia:

Aniridia is the absence of iris in the eye. Aniridia typically causes severe visual impairment; the major causative factor of this condition is foveal hypoplasia. Congenital aniridia is a rare ocular malformation that affects the development of multiple ocular structures. Aniridia is caused by a mutation in the paired box gene 6 (PAX6). A paired homeodomain transcription factor, PAX6, is a well-known regulator of eye development. Some sporadic cases have a risk of developing Wilms tumor as a part of WAGR (Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation) which is caused by deletion of both PAX6 and Wilms’ tumor gene (WT1) in the 11p13 region. PAX6 protein acts as a "master control" gene for the development of eyes and other sensory organs, certain neural and epidermal tissues as well as other homologous structures usually derived from ectodermal tissues. This transcription factor is most noted for its use in the interspecifically induced expression of ectopic eyes and is of medical importance because heterozygous mutants produce a wide spectrum of ocular defects such as Aniridia in human. Numerous PAX6 mutations have been detected in aniridia patients and premature termination of the PAX6 protein is the most frequent type of mutation. The diagnostic testing for PAX6 mutation confirms the clinical diagnosis of aniridia.

DNA sequencing facility

Retinoschisis (RS1) gene screening:

X-linked juvenile retinoschisis is an inherited eye disorder that makes the inner layer of the retina split in a spoke-wheeled pattern. It usually occurs in both eyes at once. This disease affects more males than females. Fundus examination shows areas of schisis in the macula, sometimes giving the impression of a spoke wheel pattern. Affected males typically have vision of 20/60 to 20/120. The diagnosis of X-linked juvenile retinoschisis is based on fundus findings, results of electrophysiologic testing, and molecular genetic testing. RS1 is the only gene known to be associated with X-linked juvenile retinoschisis. Retinoschisin is a cell-cell adhesion protein which helps in maintaining the integrity of the retina. Mutation in the Retinoschisin (RS1) gene results in defective protein leading to splitting of retinal layers (Schisis).Since the mode of inheritance is X-linked recessive, the genetic testing in the patient would help in assessing the carrier status, prenatal diagnosis and genetic counseling.

List of genetic tests offered by our department:
Name of the test Code No. Condition for which the test is done Specimen
RPE65 Gene Screening

L705

Retinitis Pigmentosa & Leber’s Congenital Amaurosis 10 ml of heparinized blood to be transported at room temperature
Rhodopsin (RHO) Gene Screening L724 Retinitis pigmentosa & Congenital Stationary Night Blindness
CYP1B1 Gene Screening L714 Peter’s anomaly, Congenital Glaucoma,
Primary and Juvenile Open Angle Glaucoma
MYOC Gene Screening L715 Primary and Juvenile Open Angle Glaucoma
PAX6 Gene Screening L725 Peter’s anomaly & Aniridia
Retinoschisis (RS1) Gene Screening L726 Retinoschisis
Screening the three primary mitochondrial mutations for Lebers Hereditary Optic Neuropathy (LHON) L723 Lebers Hereditary Optic Neuropathy (LHON)
BBS Genes screening for Bardet – Biedl Syndrome L722 Bardet – Biedl Syndrome