Browsing by Author "Abeysekera, H."

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Deletion analysis of the RB1 gene in retinoblastoma patients in Sri Lanka
(Faculty of Science, University of Kelaniya, Sri Lanka, 2020) Panchananthan, N.; De Silva, D.; Abeysekera, H.; Nanayakara, D.P.S.; Tirimanne, T.L.S.; Chandrasekharan, N.V.
Retinoblastoma (RB), a tumour, affecting children aged less than 5 years has a prevalence of 1 in 20,000 with twenty cases/ year predicted in Sri Lanka. Unilateral RB (60%) presents on average at 24 months and bilateral RB (40%) presents around 15 months. A family history is reported in 10%. Many cases, diagnosed late, require enucleation. Genetic testing has not been available locally, but may enable better targeting of screening for patients and their siblings and reduce the need for enucleation in affected cases. Mutations of RB1 gene lead to inactivation of pRB (retinoblastoma protein) and loss of its function. Different mutations of RB1 gene include nonsense (37%), frameshift (20%), splice site (21%), missense (5%), deletions/ duplications of one or several exons or even the entire gene (15%) and mutations in the promoter region (1%). Hypermethylation of the promoter region is also found in the tumours of some retinoblastoma cases. About 5-15% of retinoblastoma patients have microscopic or submicroscopic deletions, which includes the entire or substantial parts (one or several exons) of the RB1 gene. The objective of this study was to identify the presence of germline copy number variations of the RB1 gene or any of its exons in retinoblastoma patients. Primers were designed for the 27 exons and promoter region of the target gene (RB1) and a control gene (Cystic fibrosis transmembrane conductance regulator - CFTR) to compare the copy number of both genes using gene ratio analysis copy enumeration PCR (GRACE-PCR). The peak height of the melting curve was analysed for calculation of the ratio. The ratio of the peak height of the melting curve of the target (RB1) to the control gene (CFTR) was calculated for patients and normal individuals separately. A ratio of patient to normal of 1 indicates the patient is deletion negative. A ratio of 0.5 indicates a deletion and a ratio of 1.5 indicates a duplication. Seven exons (exons 3, 4, 5, 6, 8, 9 and 10) of 32 patients and 3 more exons (exons 11, 12 and 16) of 16 patients who have no germline mutation identified from targeted amplicon sequencing, were tested. One case had a deletion for all 10 exons, while one case each had deletions of exon 11 and exon 12. In conclusion, three RB cases out of 32 patients (9%) have a deletion of one or several exons which is similar to world wide data and further testing is ongoing. Genetic testing helps to determine the recurrence risk and to target intensive screening to at risk family members. This can contribute to balance the resource limited healthcare services of developing countries.
RB1 screening of retinoblastoma patients in Sri Lanka using targeted next generation sequencing (NGS) and gene ratio analysis copy enumeration PCR (GRACE-PCR)
(BioMed Central, 2023) Kugalingam, N.; de Silva, D.; Abeysekera, H.; Nanayakkara, S.; Tirimanne, S.; Ranaweera, D.; Suravajhala, P.; Chandrasekharan, V.
BACKGROUND: Retinoblastoma (RB) a tumour affecting those under 5 years, has a prevalence of 1 in 20,000, with around twenty new diagnoses per year in Sri Lanka. Unilateral and bilateral RB presents around 24 and 15 months respectively. Approximately 10% are familial. Systematic genetic testing for germline pathogenic variants of RB1, the only gene associated with an inherited risk of RB, is unavailable in Sri Lanka. Genetic testing optimizes management of affected children and at-risk siblings. This study aimed to develop accessible genetic testing to identify children with a germline pathogenic variant of RB1 in Sri Lanka. METHODS: Targeted next generation sequencing (NGS) for detecting pathogenic sequence variants and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR) for detecting RB1 copy number variations (CNVs) were performed for 49 consecutive RB patients treated between 2016 and 2020 at the designated RB care unit, Lady Ridgway hospital, Colombo. Patients (bilateral RB (n = 18; 37%), unilateral n = 31) were recruited following ethical clearance and informed consent. RESULTS: There were 26 (53%) females. Mean age at diagnosis was 18 months. Thirty-five patients (71%) had undergone enucleation. Germline pathogenic variants of RB1 identified in 22/49 (45%) patients including 18 (37%; 12 bilateral and 6 unilateral) detected by targeted NGS (2 missense, 7 stop gained, 1 splice donor, 8 frameshift variants). Six were previously undescribed, likely pathogenic frameshift variants. Four bilateral RB patients had GRACE-PCR detected CNVs including one whole RB1, two intragenic deletions (exon 12/13; exon 11 and 23) and a partial duplication of exon 27. The only familial case (affected mother and child) shared the duplication. Only 2 of 4 CNVs and 10 of 18 pathogenic variants were confirmed by whole exome sequencing and Sanger sequencing respectively, due to funding limitations. CONCLUSIONS: The study identified pathogenic or likely pathogenic germline RB1 sequence variants and copy number variants in 16/18 (89%) bilateral and 6/31(19%) unilateral cases, which is comparable to worldwide data (10-15% unilateral, 80-85% bilateral). Targeted NGS combined with GRACE-PCR significantly reduce the cost of RB1 testing in Sri Lanka, and may widen access for genetic diagnosis of RB patients in other low and middle income countries.
Retinoblastoma patients treated in Sri Lanka from 2014 to 2020: epidemiology, clinical status and correlates of lag time in seeking tertiary care services
(BioMed Central, 2024) Kugalingam, N.; De Silva, D.; Abeysekera, H.; Nanayakkara, S.; Tirimanne, S.; Chandrasekharan, V.; Jayawardana, P.L.
BACKGROUND Retinoblastoma (RB) is a tumour of children < 5 years with a incidence of 1 in 20,000. Around 20 RB cases are diagnosed yearly in Sri Lanka, a lower middle-income country with high literacy levels and healthcare free at point of delivery. Incidence, local and systemic severity and mortality related to RB are reportedly high in low- and middle- income countries in comparison to higher income countries. Aims of this study were to describe demographic, socioeconomic, and clinical characteristics of Sri Lankan RB patients attending the designated RB unit at the Lady Ridgeway Hospital (LRH), Colombo between January 2014 to December 2020, and determine correlates of lag time (LT) for first tertiary care visit after detecting the first symptom/sign.METHODS Two descriptive cross-sectional studies (DCSS) were conducted, one on 171 RB patients with demographic and clinical data collected between 2017 and 2020. In 2021, the second DCSS took place where socioeconomic and further demographic data were collected using telephone interviews, recruiting a subgroup of 90 (53%), consenting and contactable RB patient/ parent pairs. Bivariate and multivariable analyses were applied to determine correlates of LT of > 4 weeks for first tertiary care visit. Results were expressed as odds ratios and 95% confidence intervals.RESULTS LRH survey (N = 171): Median age at diagnosis was 15 months (range 1-94 months; IQR: 8-27); 89 (52%) were females. Groups D and E tumours were 25.7% (n = 44) and 62.6% (n = 107) respectively with 121 (71%) enucleations. The number of deaths were 2 (1.2%). Telephone survey (N = 90): Proportion with LT of > 4 weeks for first tertiary care visit was 58% (n = 52). None of the putative risk factors (ethnicity, parental educational level, socioeconomic status, distance from residence to tertiary care unit and receiving financial assistance) were associated with LT in both analyses.CONCLUSION Despite a high proportion with groups D and E tumours and enucleations, mortality rate was low, most likely due to availability of designated tertiary care. No correlates for LT of > 4 weeks for tertiary care presentation were identified. Early RB detection needs rigorous implementation of screening strategies and increased awareness among primary care health workers and parents.