Browsing by Author "Atapattu, N."

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Development and evaluation of a cost effective method for the molecular diagnosis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) in Sri Lanka.
(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Panchananthan, V.; De Silva, D.; Rathnayake, P.; Atapattu, N.; Chandrasekharan, N.V.
Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are genetic disorders caused by imprinting defects on chromosome 15q11-q13. Prader –Willi syndrome is caused by loss of expression of some paternal genes and Angelman syndrome is caused by loss of expression of some maternal genes on chromosome 15q due to a deletion or uniparental disomy (UPD) of chromosome 15q or methylation centre defects in this region. The methylation specific PCR assay can detect ~99% of cases of PWS caused by deletion or UPD but can miss the 1% of cases caused by imprinting centre defects. The methylation specific PCR can detect around 90% of cases of AS caused by a deletion or UPD but can miss the 10% of cases caused by UBE3A mutation and imprinting centre defects. Objectives of this study were to establish diagnostic testing for PWS and AS among Sri Lankan patients using methylation specific PCR and to develop an in house DNA modification method for use in the methylation specific PCR assay. Forty three patients (suspected PWS = 24, AS =19) were recruited after ethical clearance and informed consent from parents. DNA was extracted from blood and methylation specific PCR (MS-PCR) performed using control primers (SNRPNF and SNRPNR), maternal (MF1 and MR1) and paternal (PF2 and PR2) primers after the modification of DNA using a sodium bisulfite modification kit while some of the samples were modified using a modified protocol established in our laboratory. Eleven out of twenty four suspected PWS cases and none of the nineteen suspected AS cases were positive on testing. The kit based modification generated consistent reliable results while the in house method requires further optimization. In conclusion, the methylation specific PCR was successful in detecting methylation abnormalities associated with PWS and AS and is a potentially useful test to confirm or refute the diagnosis of suspected cases. The MS-PCR negative, suspected AS cases merit clinical review to determine the need for mutation testing prior to exclusion of AS.
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.
IPEX syndrome with membrano-proliferative nephrotic syndrome
(Sri Lanka Medical Association, 2013) Rodrigo, R.; Atapattu, N.; de Silva, K.
No Abstract Available