Biju Joseph

Tumor necrosis factor allelic polymorphism with diabetic retinopathy in India

The association of tumor necrosis factor (TNF) with diabetic retinopathy (DR) has been described previously. A total of 207 Asian Indian patients of 15-year duration of type 2 diabetes were identified. This group included (i) 100 patients with DR and (ii) 107 patients without retinopathy (DNR). In this study, we correlated the length of the (GT)n microsatellite di-nucleotide repeat upstream to the promoter region of TNF gene with susceptibility for the development of retinopathy. The microsatellite was polymerase chain reaction amplified and electrophoresed on polyacrylamide gel and silver stained. In our study population, there were 18 alleles ranging from 97 to 131 base pairs (bp). Allele 4 (103 bp) had a higher prevalence (9.81%) in the DNR group compared to that (2.5%) in the DR group (P=0.002). Patients with retinopathy and allele 8 (111 bp) had a tendency to develop proliferative diabetic retinopathy (PDR). In this study of Indian subjects, it is suggested that allele 4 is a low risk allele for developing retinopathy and allele 8 (111 bp) shows an association with PDR.

Consanguinity and Ocular Genetic Diseases in South India: Analysis of a Five-Year Study

Objective: Consanguineous marriage is a widely practised social custom in Asia and northern Africa. In south India, Dravidian Hindus have contracted consanguineous marriages for over 2,000 years. In the present study, the influence of consanguinity on the prevalence of visual disorders was examined in patients attending a specialist genetic eye clinic. Subjects and Methods: A total of 2,335 patients attending Sankara Nethralaya, Chennai, India, were screened for genetic eye disorders over a five-year period. The patients were drawn from all parts of India and from neighbouring countries in south Asia. Results and Discussion: Six hundred and seventy-three (28.8%) of the patients tested for ophthalmic genetic disorders reported a family history of consanguinity. The majority (n = 574) of these families were from south India. In the patient group as a whole, the most common form of consanguineous union was between first cousins (n = 367), followed by uncle/niece marriage (n = 177), equivalent to a mean coefficient of inbreeding α = 0.0202. Among the consanguineous families, 430 of 673 (63.9%) had retinitis pigmentosa, 167 of these cases were autosomal recessive and 199 were isolated cases. The public in regions such as south India should be made aware of the merits and demerits of consanguineous marriages.

Methylation status of RB1 promoter in Indian retinoblastoma patients.

Retinoblastoma can arise due to mutational inactivation or methylation of RB1 gene promoter. A 600-bp CpG island consisting of the essential promoter is present at the 5’ end of RB1 gene. Hypermethylation of the CpG island within the RB1 promoter region has been described in unilateral retinoblastoma. Invitro and invivo studies have suggested that methylation of the RB1 promoter dramatically reduces gene activity. In the present study methylation status of the CpG island within the promoter region of RB1 gene has been evaluated by methylation specific polymerase chain reaction to define the molecular mechanism responsible for retinoblastoma in Indian patients. One unilateral and two bilateral non-hereditary patients had methylation of the RB1 promoter region in which 6.6 % of our patients had complete methylation of the RB1 promoter region. This study shows methylation of RB1 promoter is not a major mechanism for retinoblastoma patients in India. Methylation analysis is used in genetic counseling of the family.

RPE65 gene: multiplex PCR and mutation screening in patients from India with retinal degenerative diseases

We used multiplex PCR followed by sequencing to screen for mutations in the 14 exons of theRPE65 gene in early-hildhood-onset autosomal recessive retinitis pigmentosa (arRP) and Leber’s congenital amaurosis (LCA) patients. The RPE65 protein is believed to play an important role in the metabolism of vitamin A in the visual cycle and mutations identified in the gene could have implications for vitamin A-based therapeutic intervention. We were able to identify a homozygous mutation (AAT → AAG) in exon 9 in an arRP patient and a heterozygous missense transversion (AAT → AAG) also in exon 9 of an LCA patient. We also identified a polymorphism in exon 10 (GAG → GAA) in an arRP as well as an LCA patient. Mutation screening would be greatly facilitated by multiplex PCR which could cut down costs, labour and time involved. The nucleotide changes observed in this study could bede novo. Though a larger study has been undertaken, from the preliminary results it appears that in India theRPE65 gene seems to be less involved in causation of LCA.

Molecular-genetic analysis of two cases with retinoblastoma: benefits for disease management

Effective counselling and management of retinoblastoma families using genetic information is presently practised in many parts of the world. We studied histopathological, chromosomal and molecular-genetic data of two retinoblastoma patients from India. The two patients, one with bilateral and the other with unilateral retinoblastoma, underwent complete ophthalmic examination, cytogenetic study, retinoblastoma gene (RB1) mutational analysis andRB1 promoter region methylation screening. In the bilateral retinoblastoma patient deletion of chromosome region 13q14 in peripheral blood lymphocytes and a hemizygous novel 8-bp deletion in exon 4 ofRB1 in tumour sample were observed. In the unilaterally affected patient CGA to TGA transition protein truncation mutations were observed in exons 8 and 14 ofRB1.

CGA codons multiplex PCR in rapid diagnosis of retinoblastoma

Background: Multiplex polymerase chain reaction allows amplification of multiple target sequences of a genome under identical conditions in a single tube. This one-shot polymerase chain reaction detection is time and cost effective when large or multiple genes, with many target fragments are investigated. This is applicable for retinoblastoma susceptibility gene having 27 exons with recurrent mutations reported at most of the 12 CGA codons. Materials and Methods: Multiplex polymerase chain reaction assay for the amplification of 12 CGA codons, which constitutes about 50 % of retinoblastoma susceptibility gene mutations has been designed. The time and cost (includes only reagent cost) involved in both multiplex and uniplex polymerase chain reaction was also calculated. Results: Twelve CGA codons were multiplexed in 5 instead of 12 uniplex polymerase chain reactions, which took 36 hours and 9.78 US

Retinoblastoma: genetic testing versus conventional clinical screening in India.

whereas multiplex polymerase chain reaction took 15 hours and 6.88 US

Retinoblastoma: A Diagnostic Model for India

. Multiplex polymerase chain reaction method saved 58.3% of time and 29.6% of cost over uniplex polymerase chain reaction. Conclusion: Saving time by more than half and cost by nearly a third would help clinicians and geneticists while counseling retinoblastoma patients.