Development of a Low Cost Semiquantitative Polymerase Chain Reaction Assay for Molecular Diagnosis of Williams Syndrome.

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.

RB1 screening of retinoblastoma patients in Sri Lanka using targeted next generation sequencing (NGS) and gene ratio analysis copy enumeration PCR (GRACE-PCR).

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.