IERG Abstracrt Book.indd - LV Prasad Eye Institute

Oral Presentation31This domain has a conventional sequence of AB type arrangement of Greek key motif typicalof vertebrate crystallins and is also closely similar to the β γ-crystallin domains of AIM1 insequence. Though this domain structurally resembles the eye lens g-crystallin, it has a relativelymoderate thermal (49°C, H 1.29 x 104 ± 216 kJ mol-1) and equilibrium stability of C1/2,[GdmCl] of 1.14 M. Brainillin domain upon unfolding retains significant tertiary structure with aconsiderable loss of secondary structure. During the early equilibrium unfolding (at sub-molarconcentrations of GdmCl), the protein is precipitated indicating the aggregation of partiallyunfolded species, a phenomenon exhibited by a cataract-related mutants of γ-crystallin.Conclusions: This study provides further insight on the sequence-structure relationship ofvarious β γ-crystallin domains, and would be used to explore the structural effects of cataractrelatedmutations seen in lens crystallins. The fact that β γ-crystallin domains are also the partof diverse non-lens proteins, redefines the recruitment and evolution of lens crystallins.IPT 004Transcript Analysis of Constitutional Mutations in the RB1 Gene in RetinoblastomaPatients Reveals Different Patterns of MissplicingVidya Latha Parsam, 1 Chitra Kannabiran1, Mohd Javed Ali, 2 Santosh G Honavar, 2Geeta K Vemuganti 31Kallam Anji Reddy Molecular Genetics Laboratory, 2 Ocular Oncology Service, 3 OphthalmicPathology Service L V Prasad Eye Institute, Hyderabad, India.Purpose: RNA from the blood of 16 retinoblastoma (Rb) patients was analyzed a) tocharacterize the effects of mutations detected in genomic DNA including consensus splicesite mutations, other exonic substitution mutations or deletions of exons, and b) to identifymutations in cases where no mutations were detectable in genomic DNA.Methods: Total RNA from fresh blood of all the patients and available family members wasisolated using Trizol reagent and first strand cDNA synthesis was prepared using oligo dT.Complete RB1 cDNA was amplified by RT-PCR and was further analyzed to characterize theabnormal transcripts.Results: Transcript analysis of 2 splice site mutations, IVS22+5 G>C and IVS11-1 G>Aidentified in genomic DNA of 2 patients, revealed single exon skipping in both cases.A missense substitution of p.Leu218Val in exon 7 found in a proband with bilateral Rb resultedin two abnormal transcripts. In 2 probands with no mutations identified in genomic DNA,RNA analysis was informative- one patient had a deletion of exon 6 and the 2nd patient hadmore than one aberrant transcript involving exons 21 and 22. Deletions of exons 23-25 and ofexon 14 identified by quantitative multiplex PCR of genomic DNA in a two familial cases wereconfirmed by RNA analysis.Conclusions: RNA analysis revealed the effects of splice mutations, as well the splicing defectdue to a missense substitution. Our study also demonstrates the utility of mRNA screening toenhance detection of mutations in cases with no identifiable mutations in genomic DNA.