Delineation of chromosome abnormalities in Thai patients by chromosome microdissection

Abstract

Chromosome microdissection and in vitro amplification of dissected chromosomal fragments, followed by labeling and fluorescence in situ hybridization (FISH) to normal metaphase chromosomes or “micro-FISH” can be used to identify marker chromosomes for both prenatal and postnatal cytogenetic analysis. The primary objective of this study is to identify the origin of marker chromosomes in three Thai patients with chromosomal disorders. Secondary objective is to apply this method for routine laboratory practice. The method involves the microdissection of five to ten fragments of the related chromosomes from a GTG-banded metaphase spread. Then, the dissected chromosomal fragments were amplified using degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The PCR products were labeled by PCR with digoxigenin-11-dUTP. Digoxigenin-labeled probes were detected with mouse anti-digoxigenin and the hybridization signal was detected by anti-mouse immunoglobulin conjugated with Alexa 488. We demonstrated the advantage of this approach in routine clinical cytogenetic testing for the analysis of cases involving a translocation, an insertion or a marker chromosome with an additional material of unknown origin. The micro-FISH probe was used successfully to determine the X and 1 translocation chromosome unidentifiable by conventional cytogenetic (GTG-banded) analysis. This study could not exactly detect the band that resulted from duplication of itself. For conclusion, micro-FISH technique provides a possibility to determine the origin of unbalanced chromosomal rearrangements.

Keywords: Chromosome painting, Micro-FISH, Chromosome microdissection, DOP-PCR, Chromosomal marker