Dr. RACHNA MEEL

DR. SEEMA KASHYAP, Dr.SAHIL AGRAWAL, Dr. MANDEEP SINGH BAJAJ

Abstract

Purpose:To isolate cell free DNA(cfDNA) from aqueous humor(AH) in Retinoblastoma(Rb) eyes & validate its use for genetic studies.

Methods:AH was obtained in 26 eyes undergoing enucleation(Group A) or intravitreal chemotherapy(Group B). Isolation of cfDNA was done using QIAamp® Circulating Nucleic Acid kit & utilized for next generation sequencing(NGS).

Results:We could isolate cfDNA in all eyes(72% bilateral & 28% unilateral) with distribution peak between 140-160 bp & mean concentration of 14ng/µl for Group B & 27.75ng/µl for Group A. NGS done on 4 samples showed Rb1 gene mutations – Inframe deletion(c.78-80del, p.Pro29del), nonsense(c.2236G>T,p.Glu746Ter), silent(c.30C>T,p.Ala10=),missense(c.59C>T, p.Pro20Leu) & intronic(c.1422-8del;c.2212-9C>A) to be most common. Genetic profile of cfDNA extracted from AH & genomic DNA from tumor tissue was comparable.

Conclusion:This is the first report from the Indian subcontinent on isolation and genetic analysis of cfDNA from AH of Rb eyes.

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