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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty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 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.Item Molecular diagnosis of velo-cardio-facial syndrome among sri lankan patients with congenital cardiac defects(Sri Lanka College of Paediatricians, 2015) Tevarajan, I.; Ranaweera, D. M.; Perera, S.; Samarasinghe, D.; Morawakkorala, R.; Silva, R. L.; de Silva, D.; Chandrasekharan, N.V.Velo cardio facial Syndrome (VCFS) is caused by a 3 Mb deletion of chromosome 22qll.2. Its multiple clinical features include orofacial clefting, congenital cardiac defects (especially conotruncal),developmental delay and learning difficulties. Hypoparathyroidism and thymic hypoplasia are associated. Dysmorphic features include expressionless face, prominent nose, narrow eyes and long fingers/ toes. Clinical diagnosis is difficult due to its variability making molecular diagnosis essential but this is often too expensive for widespread use. We have developed a less expensive semi-quantitative PCR method for diagnosing VCFS and report preliminary results in congenital cardiac defect patients.OBJECTIVE: • Identify the 22qll.2 deletion syndrome among a selected group of children with typical cardiac defects • Describe clinical features of affected cases DESIGN, SETTING AND METHOD: TweIve children (6 males, mean age 3y lmo) with conotruncal congenital cardiac anomalies or cardiac defects associated with other clinical feature of VCFS were .recruited following informed consent from parents. Ethical approval had been granted for this study. A blood sample was obtained for DNA extraction and the clinical data recorded. Molecular diagnosis was performed using semi-quantitative PCR. RESULTS: Three cases were positive for the deletion. Their cardiac anomalies were an interrupted aortic arch,tetralogy of Fallot and right sided aortic arch. None had palatal anomalies and two (67%) had learning difficulties. None had a positive family history. Only one had facies that were typical. The negative cases included six with aortic arch anomalies, none with clefting and 4 with learning difficulties(44). Two had a family history suggestive of VCFS and two had typical facial features. CONCLUSIONS: Three out of the 12 children were positive for the 22qll.2 deletion.Item Functional analysis of a novel parasitic nematode-specific protein of Setaria digitata larvae in Culex quinquefasciatus by siRNA mediated RNA interference(BioMed Central, 2018) Somarathne, M.B.C.L.; Gunawardene, Y.I.N.S.; Chandrasekharan, N.V.; Ellepola, A.N.B.; Dassanayake, R.S.BACKGROUND: Functional analysis of animal parasitic nematode genes is often quite challenging due to the unavailability of standardised in vitro culture conditions and lack of adequate tools to manipulate these genes. Therefore, this study was undertaken to investigate the suitability of Culex quinquefasciatus, as an in vivo culture platform for Setaria digitata larvae and RNA interference (RNAi), as a post-transcriptional gene silencing tool to study the roles of a vital gene that encodes a novel parasitic nematode-specific protein (SDNP). RESULTS: The red colour fluorescence detected following RNAi injection to the thorax of C. quinquefasciatus indicated the uptake of dsRNA by S. digitata larvae. The reduction of SDNP transcripts in siRNA treated larvae compared to non-treated larvae, as determined by qPCR, indicated that the siRNA pathway is operational in S. digitata larvae. The observation of motility reductions and deformities during the development indicated the association of SDNP in larvae locomotion and development processes, respectively. The irregularities in the migration of larvae in mosquitoes and elevated survival rates of mosquitoes compared to their untreated counterparts indicated reduced parasitism of S. digitata larvae in mosquitoes upon targeted downregulation of SDNP by siRNA treatment. CONCLUSION: SDNP plays vital roles in muscle contraction, locomotion, development processes, larval development and parasitism of S. digitata. Its ubiquitous presence in parasitic nematodes and its absence in their hosts provide a tantalising prospect of the possibility of targeting SDNP for future development of anthelmintic drugs. The susceptibility of the larval stages of S. digitata for RNAi in Culex quinquefasciatus was also demonstrated for the first time in this study.Item Development of siRNA mediated RNA interference and functional analysis of novel parasitic nematode-specific protein of Setaria digitata(Academic Press, 2018) Somarathne, M.B.C.L.; Gunawardene, Y.I.N.S.; Chandrasekharan, N.V.; Dassanayake, R.S.Despite the differences of the host, parasitic nematodes may share commonalities in their parasitizing genes. Setaria digitata novel protein (SDNP) is such an entity which is parasitic nematode-specific and having sequence similarities with those of W. bancrofti, B. malayi, Loa loa and Onchocerca volvulus. Post-transcriptional gene silencing by siRNA mediated RNA interference (RNAi) is a widely used technique in functional genomics. Though the technique has been used in several free-living, plant and animal parasitic nematodes, it has not yet been tried out for the filarial worm S. digitata. In this study, we developed an effective siRNA delivery method by microinjection and utilized the siRNAi tool to knockdown SDNP to study the phenotypic and cellular changes associated with the interference. qPCR analysis revealed, a significant reduction of SDNP transcript levels following siRNA microinjection into S. digitata adult worms. Similarly, immunohistochemical staining indicated a reduction of SDNP protein expression. Furthermore, worms treated with siRNA showed a significant reduction of microfilariae release together with embryonic lethality by arresting an early developmental stage compared to non-treated worms. A distinct motility reduction was also observed in treated worms compared to non-treated counterparts. This is the first report of the amenability of S. digitata to the siRNA induced RNAi. The presence of inter-domain linkers of muscle-specific twitchin kinase and calcium-dependent protein kinase isoform CDPK1 together with what our results revealed suggest that SDNP is most likely a protein involved in muscle movement and growth and development of the nematode. Hence SDNP has the characteristics of a potential drug target.Item Identification of cattle, buffaloes and rodents as reservoir animals of Leptospira in the District of Gampaha, Sri Lanka(Biomed Central, 2017) Denipitiya, D.T.H.; Chandrasekharan, N.V.; Abeyewickreme, W.; Hartskeerl, R.A.; Hapugoda, M.D.BACKGROUND: Leptospirosis is an important emerging infectious disease in Sri Lanka. Rats are the most important reservoir of Leptospira but domestic and wild mammals may also act as important maintenance or accidental hosts. In Sri Lanka, knowledge of reservoir animals of leptospires is poor. The objective of this study was to identify potential reservoir animals of Leptospira in the District of Gampaha, Sri Lanka. FINDINGS: Blood and kidney samples were collected from 38 rodents and mid-stream urine samples were randomly collected from 45 cattle and five buffaloes in the District of Gampaha. Kidney and urine samples were tested by real-time polymerase chain reaction (PCR) and serum samples were tested by the microscopic agglutination test (MAT). Of the 38 rodent kidney samples, 11% (4/38) were positive by real-time PCR. The prevalence of leptospiral carriage was 11% (3/26) and 8% (1/12) in female and male rodents, respectively. Three rodent serum samples were positive by MAT. Of the 50 cattle/buffalo urine samples tested, 10% (5/50) were positive by real-time PCR. The prevalence of leptospiral carriage was 9% (4/45) and 20% (1/5) in cattle and buffaloes, respectively. CONCLUSION: Results of PCR and MAT showed that Leptospira were present in a significant proportion of the rodents and farm animals tested in this study and suggest that these (semi-) domestic animals form an infection reservoir for Leptospira. Therefore, there is a potential zoonotic risk to public health, most notably to farmers in this area.Item Modified mismatch polymerase chain reaction-restriction fragment length polymorphism detected mutations in codon 12 and 13 of exon 2 of K-ras gene in colorectal cancer patients and its association with liver metastases: Data from a South Asian country(Medknow Publications, 2016) Faleel, F.D.; Zoysa, M.I.; Lokuhetti, M.D.; Gunawardene, Y.I.N.S.; Chandrasekharan, N.V.; Dassanayake, R.S.AIM: Mutations in K-ras codon 12 and 13 of exon 2 are known to affect prognosis and impart resistance to anti-epidermal growth factor monoclonal antibody therapy in colorectal carcinoma (CRC). Our aim was to investigate the utility value of modified mismatch polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to detect mutation in K-ras codons of CRC patients and to relate the mutational status to liver metastasis. METHODOLOGY: Mismatch PCR-RFLP was developed to detect K-ras mutations in DNA isolated from paraffinized tumor tissue of thirty CRC patients. All patients had 5 year follow-up data to detect liver metastasis. Cross-tabulations were generated between K-ras mutations and the metastatic status. The Chi-square test was used to indicate statistical significance of the association. RESULTS: Of the 30 CRC patients investigated, K-ras mutations of codons 12 and/or 13 of exon 2 were detected in 14 (46.6%). Meanwhile, 13 patients (43.3%) were observed to have developed liver metastases. There was a significant association between the presence of the K-ras mutation in codon 12 and the occurrence of liver metastasis (χ2 = 4.693, P = 0.030) on the contrary to the mutation in codon 13 to which such occurrence of liver metastases was not seen (χ2 = 1.884, P = 0.169). CONCLUSION: Codon 12 of exon 2 of K--ras gene detected by modified mismatch PCR-RFLP assay is significantly associated with liver metastasis in CRC patients during the first 5 years after surgery. Thus, modified mismatch PCR-RFLP protocol is a suitable method in this setting to detect K-ras gene mutations predicting liver metastasis in CRC patients.Item Application of a real time Polymerase Chain Reaction (PCR) assay for the early diagnosis of human leptospirosis in Sri Lanka(Academic Press, Elsevier, 2016) Denipitiya, D.T.H.; Chandrasekharan, N.V.; Abeyewickreme, W.; Hartskeerl, C.M.; Hartskeerl, R.A.; Jiffrey, A.M.; Hapugoda, M.D.Leptospirosis has a major impact on health in Sri Lanka but is probably grossly under-recognized due to difficulties in clinical diagnosis and lack of diagnostic laboratory services. The objective of this study was to establish and evaluate a SYBR Green-based real-time Polymerase Chain Reaction (rt-PCR) assay for early, rapid and definitive laboratory diagnosis of leptospirosis in Sri Lanka. The rt-PCR assay was established and analytical specificity and sensitivity were determined using reference DNA samples. Evaluation of the assay for diagnosis of clinical samples was performed using two panels of serum samples obtained from 111 clinically suspected adult patients. Patients were confirmed as leptospirosis (n = 65) and non-leptospirosis (n = 30) by the Patoc - MAT. Other 16 samples gave ambiguous results. The analytical sensitivity of the rt-PCR was approximately 60 genome copies and no cross-reactivity was observed with saprophytic Leptospira spp. and other pathogenic microorganisms. Based on confirmation with Patoc-MAT on paired samples this corresponds to a diagnostic sensitivity and specificity of 67.7% (44/65) and 90.0% (27/30), respectively. This study showed that rt-PCR has the potential to facilitate rapid and definitive diagnosis of leptospirosis during early phase of infection in Sri Lanka.
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