Abstract:
Velocardiofacial Syndrome is associated with orofacial clefting, congenital heart defects,
and developmental delay. It is caused by a 3Mb deletion of chromosome 22q11.2. The
gold standard test is FISH but using commercial probes incurs high costs which are
beyond the means of most patients. This study was aimed at developing a FISH based
assay. Bacterial Artificial Chromosome (BAC) clones (FISH mapped and end-sequenced)
spanning a region within the deleted and control regions were identified using publicly
available databases and obtained from BACPAC Resource Center, Children’s Hospital
Oklahoma, USA. Bacterial cultures of the BAC clones, RP11-1057H19 (173,947bp) for
the target region (22q11.2), and CH17-338N2 (205,938bp) for the control region
(22q13.3) were grown and plasmid DNA was prepared using optimized methods. The
isolated BAC DNA was further confirmed by PCR using 3 sets of primers spanning the
two regions. BAC DNA (target and control) were fluorescently labelled by degenerate
oligonucleotide PCR (DOP- PCR) using Fluorescien-12-dUTP (Green) and Chroma Tide
Alexa Fluor 546-14-dUTP (Orange) respectively. The labeled DNA was purified by size
exclusion chromatography and a small aliquot run on an agarose gel and visualized. If
necessary, the labeled DNA fragments were DNase treated using an optimized protocol to
obtain labeled fragments in the range of 200-500bp. The labeled DNA probes were used
to perform FISH analysis of prepared metaphase spreads from three patients whose
samples had already been tested using a validated, commercial probe. One patient had a
deletion in the target region while two were negative. These results were in agreement
with those obtained using commercial probes. The developed FISH probe can distinguish
deleted from non-deleted cases. The preliminary data supports this as a useful diagnostic
test for VCFS. Analysis of more samples is ongoing.
Keywords: Fluorescent in situ Hybridization, Velocardiofacial syndrome.
