Medicine
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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty
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Item Development of a TaqMan-based dosage analysis PCR assay for the molecular diagnosis of 22q11.2 deletion syndrome(Genetics Society of Japan, 2025-01) 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.A hemizygous 1.5-3.0-Mb microdeletion of human chromosome 22q11.2 with the loss of multiple genes including histone cell cycle regulator (HIRA) causes 22q11.2 deletion syndrome (22q11.2 DS), a common disorder with variable manifestations including congenital malformations affecting the heart, palate and kidneys in association with neurodevelopmental, psychiatric, endocrine and autoimmune abnormalities. The aim of this study was to develop a TaqMan-based dosage analysis PCR (TaqMan qPCR) for use as a rapid, cost-effective test for clinically suspected patients fulfilling previously described criteria for molecular diagnosis of 22q11.2 DS in a lower middle-income country where the cost of testing limits its use in routine clinical practice. Nineteen patients were recruited with informed consent following ethical approval from the Ethics Review Committee, Lady Ridgeway Hospital for Children, Colombo. Dosage analysis of extracted DNA was performed using a TaqMan qPCR assay by amplifying regions within the target (HIRA) and control (testin LIM domain protein (TES)) genes of suspected patient (P) and unaffected person (N) samples. For detection of a deletion, the normalized value (HIRA/TES dosage) of a P sample was compared with that of an N sample. A ratio of P:N of 0.5 confirmed the presence of a deletion while a ratio of 1.0 refuted this. Seven of the 19 patients were found to have a HIRA deletion, confirming the diagnosis of 22q11.2 DS, with these results being in complete agreement with those of fluorescence in situ hybridization (FISH) (performed in nine of the 19 cases) and whole-exome sequencing (all 19 samples tested). This TaqMan qPCR assay was able to reliably distinguish HIRA-deleted cases from non-deleted ones. The assay was both cheaper and faster compared to commercially available alternatives in our setting, including FISH and multiple ligation-dependent probe amplification.Item Evaluation of an In-House genetic testing method for confirming Prader-Willi and Angelman Syndromes in Sri Lanka(Clinical Laboratory Publications, 2024) Kugalingam, N.; De Silva, D.; Rathnayake, P.; Atapattu, N.; Ranaweera, D.M.; Chandrasekharan, N.V.BACKGROUND Prader-Willi syndrome (PWS, MIM 176,270) and Angelman syndrome (AS, MIM 105,830) are caused by imprinting defects of chromosome 15q11-13, with loss of maternal gene expression causing AS and paternal gene expression causing PWS. The diagnosis, once established in most cases by using a methylation-specific PCR test, enables appropriate therapeutic interventions and avoids the need for further investigations. Genetic testing for PWS/AS is limited in Sri Lanka (and in other low- and middle-income countries), mainly because parents are unable to pay for testing as these are not funded by the health service.METHODS Ninety cases (46 female) with clinical features suggesting PWS (n = 37) and AS (n = 53), referred by a pediatric endocrinologist and a pediatric neurologist, were recruited. Clinical information and blood samples were obtained following informed consent. DNA was extracted and methylation-specific PCR (MS-PCR) was performed following bisulfite modification of DNA by using an in-house method and a kit. Results were validated using known positive controls. Parent-child trio DNA samples were used in cases with confirmed PWS and AS to determine if the disease was due to a deletion or uniparental disomy. The cost of the MS-PCR testing of the two modification methods and the microsatellite analysis was determined.RESULTS Among the suspected PWS cases, 19/37 were positive, while 5/53 of the suspected AS cases were positive. The lower identification rate of AS is probably related to the overlap of clinical features of this condition with other disorders. The kit-based modification method was more reliable, less time-consuming, and cost-effective in our laboratory.CONCLUSIONS The kit-based modification followed by MS-PCR described in this study enables more affordable genetic testing of suspected PWS/AS cases, and this is likely to improve patient care by targeting appropriate therapy for the affected cases. Parental genetic counselling is made possible regarding the low recurrence risk, especially where a deletion or uniparental disomy is confirmed. In MS-PCR, negative cases with a strong clinical suspicion of AS, UBE3A mutation testing is required. In addition, imprinting center mutation/deletion testing may also be needed in strongly clinically suspected, MS-PCR negative PWS and AS cases.Item 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.Item Genome organization, in-silico structure, and cellular localization of putative lipid transporter, ARV1 from parasitic nematode Setaria digitata(Elsevier Inc., 2022) Wickramatunga, P.G.T.S.; Gunawardene, Y.I.N.S.; Chandrasekharan, N.V.; Dassanayake, R.S.Setaria digitata, a nematode that lives in the peritoneal cavity of ruminants is the causative agent of cerebrospinal nematodiasis affecting livestock health. The ‘ACAT related enzyme 2 required for viability 1’ (arv-1) gene encodes putative lipid transporter that is essential in eukaryotes. The molecular characterization of nematode arv-1 has scarcely been studied and putative arv-1 isolated from S. digitata was used for this purpose. Docking and computer simulation studies using a modeled 3D structure of S. digitata ARV1 (Sd-ARV-1) with ceramide ligands revealed that the amino acid residues, Ile182, Leu56, Ala61, Gln186 and Gln146 are likely involved in the formation of potential sphingolipid binding sites having the same conserved residues in other nematodes. Sd-arv-1, a single copy gene, genomic region (1676 bp) had five exons encoding 217 amino acids, being interspersed by four introns showing a similar gene organization to other nematodes. Sd-ARV-1 is expressed ubiquitously at all development stages of the S. digitata life cycle. Tissue localization analyses revealed that Sd-ARV-1 was significantly expressed in the longitudinal muscle layer, endodermis, uterine wall, eggs, growing embryos inside the uterus, microfilariae, intestinal wall, esophagus lumen, dorsal nerve cord and ventral nerve cord. Therefore, ARV1 is a structurally conserved, ubiquitously expressed protein, which may be involved in development, reproduction, tissue remodeling, muscle contraction etc., in nematodes.Item Reconstruction of Metabolic Pathways for the Setaria digitata Whole Genome(Sri Lanka Medical Association, 2020) Rashanthy, N.; Kothalawala, M.S.A.; Mugunamalwaththa, T.S.; Darshika, W.A.S.; Lakmali, G.L.Y.; de Zoysa, K.; Chandrasekharan, N.V.; Gunawardene, Y.I.N.S.; Suravajhala, P.; Dassanayake, R.S.INTRODUCTION AND OBJECTIVES: Setaria digitata is a Wolbachia-free filarial parasite that resides in the abdominal cavity of ungulates. It can cause cerebrospinal nematodiasis (CNS) in unnatural hosts such as sheep, goats, which causes a serious threat to livestock farming. Furthermore, S. digitata can also infect humans causing several conditions showing a gradual adaption to humans. METHODS: Despite, to date, complete a metabolic pathway reconstruction of S. digitata has not been undertaken and therefore, in this study the latter analyses were carried out using BLAST2GO software. RESULTS: Metabolic pathway analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified 111 enzymes found in total of 246 contigs that involve in 95 metabolic pathways, in which the most over-represented pathways are Biosynthesis of antibiotics, Phosphatidylinositol signaling system and Purine metabolism. Since S. digitata does not harbor Wolbachia endosymbiont, it was theorized that the S. digitata genome must encodes genes to carryout haem, riboflavin and nucleotides pathways, otherwise encoded by Wolbachia genome, potentially through lateral transfer of Wolbachia to an ancestor of S.digitata. Here, KEGG analysis identified 16 enzyme coding genes involve in nucleotide biosynthesis and one enzyme involve in riboflavin biosynthesis pathway. Although studies have revealed that FAD and glutathione pathways are complete in all nematode genomes, the genes encoding FAD and glutathione pathways were not found in the S. digitata. Moreover, complete nucleotide synthesis pathway and haem synthesis pathway were not found. CONCLUSION: This suggests that S. digitata may have evolved its own sequences to encode those biosynthetic pathways and hence calling for investigations to undertake characterization of genes involved in these pathways.