Current Pediatric Research

- Current Pediatric Research (2010) Volume 14, Issue 1

DNA damage studies in cases of Trisomy 21 using Comet Assay

Objective: To study the DNA damage levels among the cases of Down’s syndrome(DS) with that of the age and sex matched controls using single cell gel eleetrophoresis (SCGE) or Comet assay. Methods: Phytohaemagglutinin stimulated lymphocyte cell culture was car-ried out using RPMI 1640. G banded metaphase spreads obtained following harvesting was screened using automation software “Ikaros metasystem, Carl Zeiss”, Germany. Alkaline comet assay was carried out with the lymphocytes isolated using Histopaque. The lympho-cytes were layered on the agarose (NMA,LMA) coated slides. They were then subjected to alkaline medium (PH>13) followed by submarine gel electrophoresis (300mA current), neu-tralisation (Tris buffer PH-7.5), fixation and staining with silver nitrate. Comet metrics val-ues were obtained using the Comet score software. Results : All 40 cases of DS which was subjected for automation karyotyping showed pure trisomy 21 pattern. The results of Comet assay showed statistically significant elevated levels among cases as compared to con-trols. Comet length in cases was found to be 45.38 ± 12.5 μm as compared to 24.48 ± 3.2 μm in controls. Similar observations were made for comet tail length (18.35 ± 8.9 μm for cases and 2.61 ± 1.1 μm for controls) and head diameter (27.35 ± 5 μm for cases and 23.08 ± 2.6 μm for controls). Conclusion: There is increased DNA damage among the cases of Down’s syndrome which can be attributed to oxidative stress. DNA damage may be responsible for the clinical manifestations and long term outcome among the cases of Down’s syndrome.

Author(s): Jayaprakash T, Ramachandra Rao K, Vishnu Bhat B, Parkash Chand, Nandha Kumar S

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