Browsing by Author "Gunewardene, S."

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    Development of a low cost semiquantitative polymerase chain reaction assay for molecular diagnosis of williams syndrome
    (Clinical Laboratory Publications, 2024) Ranaweera, D.M.; de Silva, D.C.; Samarasinghe, D.; Perera, S.; Kugalingam, N.; Samarasinghe, S.R.; Madushani, W.Y.; Jayaweera, H.H.E.; Gunewardene, S.; Muneeswaran, K.; Gnanam, V.S.; Chandrasekharan, N.V.
    BACKGROUND: Williams Beuren Syndrome (WBS) is a well-recognized and common genetic cause of congenital heart defects, developmental delay, hypercalcemia, and characteristic facial features. It is caused by a 1.5 - 1.8 Mb heterozygous deletion of chromosome 7q11.23 with loss of around 28 coding genes. The aim of this study was to develop a low-cost, semi-quantitative PCR (sqPCR) method to detect the chromosome 7q11.23 deletion. METHODS: Twenty-four suspected WBS cases were recruited following ethical clearance and informed consent. Blood was obtained, DNA extracted and spectrophotometrically quantified using standard methods. To detect the deletion by dosage analysis, a target region within a gene located in the WBS commonly deleted region of 7q11.23 was amplified together with a control region in a duplex sqPCR assay. The control region was telomeric to the WBS commonly deleted region and was located in chromosome 7q31.2. The two target regions within the deleted region namely a locus within ELN and a marker in the intergenic region between FZD9 and FKBP6 and designated IFF, were amplified in separate duplex sqPCR assays. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene was used as the control for normalization. Included in the assay were a non-deleted and deleted individuals' samples. RESULTS: Nineteen patients were identified to have the deletion while five did not. All 24 patients' results were confirmed by whole exome sequencing and 11 also by fluorescence in-situ hybridization (FISH). CONCLUSIONS: The data obtained indicates the sqPCR assay developed in this study to be an accurate and reliable diagnostic test for WBS. Most Sri Lankan patients with WBS are diagnosed clinically, as many parents of affected WBS children are unable to afford currently available molecular diagnostic testing. This low cost sqPCR test is therefore likely to benefit Sri Lankan WBS patients, by enabling genetic testing for confirming or refuting a clinical diagnosis of WBS and may be of use in other low and middle income countries.
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    Molecular diagnosis of Williams syndrome using quantitative polymerase chain reaction (qPCR) in a cohort of Sri Lankan patients.
    (International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Ranaweera, D.M.; De Silva, D.; Panchananthan, N.; Samarasinghe, D.; Perera, S.; Morawakkorala, R.; Gunewardene, S.; Chandrasekharan, N.V.
    Williams Beuren Syndrome (WBS) is a genetic cause of congenital heart defects associated with developmental delay, hypercalcaemia and characteristic facial features. Its cause is a 1.5 to 1.8 Mb hemizygous deletion of chromosome 7q11.23 involving the loss of around 23 genes including the elastin (ELN) gene. The deletion results in copy number alterations. The aim of this study was to identify whether a group of Sri Lankan children with a clinical diagnosis of WBS could have their diagnosis confirmed or refuted by the use of genetic testing using a validated low cost method. A quantitative PCR method was evaluated for use in deletion screening. Twenty four suspected WBS cases were recruited following ethical clearance and informed consent. DNA was extracted, spectrophotometrically quantified and qPCR performed. The target used for deletion screening was the ELN gene and TES was used as the reference gene for normalization. In all assays, a 10 fold dilution series of standards, a no template control (NTC) and a negative control (NC) were included. The fold copy number change (ΔKCt) was determined and the mean for normals (n=6) was -0.087 ± 0.11 representing no loss while the mean for previously clinically diagnosed WS patients and confirmed by either Fluorescent in situ hybridization (FISH) or microarray analysis (n=6) was -1.39 ± 0.086 representing the loss of one copy (deletion). Among twenty four suspected cases, 19 (79%) had an ELN gene deletion while 5 cases did not and the findings correlated strongly with the clinical suspicions. This qPCR method was able to distinguish ELN deleted cases from the non-deleted ones. The preliminary data supports this as a useful diagnostic test for WBS. Validation of this test using FISH has been performed for five patient’s samples and the microarray confirmed positive which correlated with the qPCR results.