Singapore Undiagnosed Disease Program: Genomic Analysis aids Diagnosis and Clinical Management.

OBJECTIVE: Use next-generation sequencing (NGS) technology to improve our diagnostic yield in patients with suspected genetic disorders in the Asian setting. DESIGN: A diagnostic study conducted between 2014 and 2019 (and ongoing) under the Singapore Undiagnosed Disease Program. Date of last analysis was 1 July 2019. SETTING: Inpatient and outpatient genetics service at two large academic centres in Singapore. PATIENTS: Inclusion criteria: patients suspected of genetic disorders, based on abnormal antenatal ultrasound, multiple congenital anomalies and developmental delay. EXCLUSION CRITERIA: patients with known genetic disorders, either after clinical assessment or investigations (such as karyotype or chromosomal microarray). INTERVENTIONS: Use of NGS technology-whole exome sequencing (WES) or whole genome sequencing (WGS). MAIN OUTCOME MEASURES: (1) Diagnostic yield by sequencing type, (2) diagnostic yield by phenotypical categories, (3) reduction in time to diagnosis and (4) change in clinical outcomes and management. RESULTS: We demonstrate a 37.8% diagnostic yield for WES (n=172) and a 33.3% yield for WGS (n=24). The yield was higher when sequencing was conducted on trios (40.2%), as well as for certain phenotypes (neuromuscular, 54%, and skeletal dysplasia, 50%). In addition to aiding genetic counselling in 100% of the families, a positive result led to a change in treatment in 27% of patients. CONCLUSION: Genomic sequencing is an effective method for diagnosing rare disease or previous 'undiagnosed' disease. The clinical utility of WES/WGS is seen in the shortened time to diagnosis and the discovery of novel variants. Additionally, reaching a diagnosis significantly impacts families and leads to alteration in management of these patients.

High-throughput beta-thalassemia carrier screening by allele-specific Q-primer real-time polymerase chain reaction.

Based on a novel Q-primer real-time polymerase chain reaction (PCR) system, we designed allele-specific Q-primers for the detection of three beta-thalassemia mutations [Cd41/42(-TCTT), IVSI nt5 (G>C), and IVSII nt654 (C>T)] that have a high carrier frequency in Southeast Asia. With clear distinction between heterozygote and wild-type, DeltaC(t) (threshold cycle) values were defined. The results of evaluating 139 blinded samples by our system match perfectly with those obtained by the conventional reverse dot blot (RDB) method. With a 384-well plate that included replicates in the same analysis, our throughput reached 190 reactions per run with a turnaround time as short as 130 min, and the cost of consumables was as low as $1 (US) for each test.

Quantitative real-time polymerase chain reaction (RQ-PCR) for the rapid detection of SHOX haploinsufficiency in Leri-Weill Syndrome.

The short stature homeobox-containing (SHOX) gene, found on the human sex chromosomes, has a role in bone growth and height determination. Haploinsufficiency of the SHOX gene is believed to be responsible for poor growth such as that observed in ther Leri-Weill syndrome (LWS). This is the first report of the study of SHOX gene copy number by the technique of quantitative real-time polymerase chain reaction (RQ-PCR) in 9 patients with LWS. Only 7 patients (78%) of LWS had one copy of the SHOX gene deleted, but 2 patients (12%) have neither a single copy gene deletion nor point mutation after direct sequencing of all 7 exons. Although the majority of patients with LWS in this study have SHOX gene haploinsufficiency, there are some patients with both copies of the SHOX gene intact with absence of any point mutations in the coding region. This may be due to abnormalities in the upstream promoter, or to the effect of other candidate gene mutations. RQ-PCR is a faster and cheaper method of studying SHOX sing-copy deletions compared with the conventional fluorescence in situ hybridization (FISH), and is recommended for the detection of SHOX gene haploinsufficiency.

Systematic study on genetic and epimutational profile of a cohort of Amsterdam criteria-defined Lynch Syndrome in Singapore.

BACKGROUND: Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region. METHODS: Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed. RESULTS: Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations. CONCLUSIONS: Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.