A Pre-Leukemic DNA Methylation Signature in Healthy Individuals at Higher Risk for Developing Myeloid Malignancy.

PURPOSE: DNA methylation alterations are widespread in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS), some of which appear to have evolved independently of somatic mutations in epigenetic regulators. Although the presence of somatic mutations in peripheral blood can predict the risk of development of AML and MDS, its accuracy remains unsatisfactory. EXPERIMENTAL DESIGN: We performed global DNA methylation profiling in a case control study nested within the Singapore Chinese Health Study to evaluate whether DNA methylation alterations were associated with AML/MDS development. Targeted deep sequencing and methylated DNA immunoprecipitation sequencing (MeDIP-seq) were performed on peripheral blood collected a median of 9.9 years before diagnosis of AML or MDS, together with age-matched still-healthy individuals as controls. RESULTS: Sixty-six individuals who developed AML or MDS displayed significant DNA methylation changes in the peripheral blood compared with 167 age- and gender-matched controls who did not develop AML/MDS during the follow-up period. Alterations in methylation in the differentially methylation regions were associated with increased odds of developing AML/MDS. CONCLUSIONS: The epigenetic changes may be acquired independently and before somatic mutations that are relevant for AML/MDS development. The association between methylation changes and the risk of pre-AML/MDS in these individuals was considerably stronger than somatic mutations, suggesting that methylation changes could be used as biomarkers for pre-AML/MDS screening.

Somatic mutational landscape of hereditary hematopoietic malignancies caused by germline variants in RUNX1, GATA2, and DDX41.

Individuals with germ line variants associated with hereditary hematopoietic malignancies (HHMs) have a highly variable risk for leukemogenesis. Gaps in our understanding of premalignant states in HHMs have hampered efforts to design effective clinical surveillance programs, provide personalized preemptive treatments, and inform appropriate counseling for patients. We used the largest known comparative international cohort of germline RUNX1, GATA2, or DDX41 variant carriers without and with hematopoietic malignancies (HMs) to identify patterns of genetic drivers that are unique to each HHM syndrome before and after leukemogenesis. These patterns included striking heterogeneity in rates of early-onset clonal hematopoiesis (CH), with a high prevalence of CH in RUNX1 and GATA2 variant carriers who did not have malignancies (carriers-without HM). We observed a paucity of CH in DDX41 carriers-without HM. In RUNX1 carriers-without HM with CH, we detected variants in TET2, PHF6, and, most frequently, BCOR. These genes were recurrently mutated in RUNX1-driven malignancies, suggesting CH is a direct precursor to malignancy in RUNX1-driven HHMs. Leukemogenesis in RUNX1 and DDX41 carriers was often driven by second hits in RUNX1 and DDX41, respectively. This study may inform the development of HHM-specific clinical trials and gene-specific approaches to clinical monitoring. For example, trials investigating the potential benefits of monitoring DDX41 carriers-without HM for low-frequency second hits in DDX41 may now be beneficial. Similarly, trials monitoring carriers-without HM with RUNX1 germ line variants for the acquisition of somatic variants in BCOR, PHF6, and TET2 and second hits in RUNX1 are warranted.

Sub genomic analysis of SARS-CoV-2 using short read amplicon-based sequencing.

The novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. In this report, we present a modified sequencing workflow using short tiling (280bp) amplicons library preparation method paired with Illumina's iSeq100 desktop sequencer. We demonstrated the utility of our workflow in identifying gapped reads that capture characteristics of subgenomic RNA junctions within our patient cohort. These analytical and library preparation approaches allow a versatile, small footprint and decentralized deployment that can facilitate comprehensive genetics characterizations during outbreaks. Based on the sequencing data, Taqman assays were designed to accurately capture the quantity of subgenomic ORF5 and ORF7a RNA from patient samples and demonstrated utility in tracking subgenomic titres in patient samples when combined with a standard COVID-19 qRT-PCR assay.

A Single Amino Acid Substitution in Structural Protein VP2 Abrogates the Neurotropism of Enterovirus A-71 in Mice.

Enterovirus 71 (EV-A71) causes hand, foot, and mouth disease (HFMD) in children and has been associated with neurological complications. With no specific treatment and a monovalent vaccine limited to the Chinese market, HFMD remains a serious public health concern and an economic burden to affected societies. The molecular mechanisms underpinning EV-A71 neurovirulence have yet to be fully elucidated. In this work, we provide experimental evidence that a single amino acid substitution (I to K) at position 149 in structural protein VP2 of a non-mouse-adapted EV-A71 strain completely and specifically abrogated its infectivity in murine motor neuron-like NSC-34 cells. We showed that VP2 I149K mutant was impaired in murine SCARB2-mediated entry step but retained the ability to attach at the cell surface. In vivo, VP2 I149K mutant was fully attenuated in a symptomatic mouse model of progressive limb paralysis. While viral titers in limb muscles were comparable to mice infected with parental wild-type strain, significantly lower viral titers were measured in the spinal cord and brain, with minimal tissue damage, therefore indicating that VP2 I149K mutant is specifically impaired in its ability to invade the central nervous system (CNS). This study highlights the key role of amino acid at position 149 in VP2 in EV-A71 neurovirulence, and lends further support that the EF loop of VP2 represents a potential therapeutic target.

Variant landscape of the RYR1 gene based on whole genome sequencing of the Singaporean population.

The RYR1 gene codes for a ryanodine receptor which is a calcium release channel in the skeletal muscle sarcoplasmic reticulum. It is associated with Malignant Hyperthermia (MH) and congenital myopathies including Central Core Disease (CCD), Multiminicore Disease (MMD) and Congenital Fibre-Type Disproportion (CFTD). There is currently little information on the epidemiology of RYR1 variants in Asians. Our study aims to describe the RYR1 variant landscape in a Singapore cohort unselected for RYR1-associated conditions. Data was retrieved from the SG10K pilot project, where whole genome sequencing was performed on volunteers unselected and undetermined for RYR1-associated conditions. Variants were classified based on pathogenicity using databases ClinVar and InterVar. Allele frequencies of pathogenic variants were compared between Chinese, Indians and Malays. Using databases ExAC, GnomAD and GenomeAsia 100k study, we further compared local allele frequencies to those in Europe, America and Asia. Data was analysed using R Commander. Significant P value was set at p < 0.05. Majority of the RYR1 variants were missense mutations. We identified four pathogenic and four likely pathogenic RYR1 variants. All were related to the aforementioned RYR1-associated conditions. There were 6 carriers of RYR1 pathogenic variants amongst 4810 individuals, corresponding to an allele frequency of 0.06%. The prevalence of pathogenic variants was the highest amongst Indians (4 in 1127 individuals) (p = 0.030). Majority of pathogenic and likely pathogenic mutations were missense and located in mutational hotspots. These variants also occurred at higher frequencies in Asians than globally. This study describes the variant landscape of the RYR1 gene in Singapore. This knowledge will facilitate genetic screening for RYR1-related conditions.

Effects of acute severe acute respiratory syndrome coronavirus 2 infection on male hormone profile, ACE2 and TMPRSS2 expression, and potential for transmission of severe acute respiratory syndrome coronavirus 2 in semen of Asian men.

OBJECTIVE: To confirm if severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in semen of men with acute coronavirus disease 2019 and if their male hormone profile (testosterone, follicle-stimulating hormone, luteinizing hormone, sex hormone binding globulin, and free androgen index) is adversely affected during the acute phase of infection and any relation to the ACE2 and/or TMPRSS2 expression in human semen. DESIGN: Clinical study. SETTING: National University Hospital, Singapore. PATIENTS: Asian men aged 21-55 years who were admitted to National University Hospital, Singapore, with a laboratory-confirmed diagnosis of SARS-CoV-2 infection via nasopharyngeal swab in the acute phase of the infection, within 2-14 days of the development of symptoms or contact history, were recruited for the study. INTERVENTIONS: Blood was collected in the morning to assess the male hormone profile. Human semen were obtained by masturbation and sent to the molecular diagnostic laboratories to detect the presence of SARS-CoV-2 RNA and assess the ACE2 and TMPRSS2 expression. MAIN OUTCOME MEASURES: Male hormone profile level and expression of SARS-CoV-2 RNA, ACE2, and TMPRSS2 in human semen. RESULTS: A total of 63 men of Asian ethnicities agreed to participate in the study. Subsequently, 65% of recruited men had completely normal levels of male hormone profile. Moreover, 27% were noted to have higher luteinizing hormone levels between 6.6 and 16.1 IU/L (normal range, 0.8-6.1 IU/L), and 10% had higher follicle-stimulating hormone levels between 13.6 and 41.6 IU/L (normal range, 1.5-12.4 IU/L); all had normal testosterone levels. No SARS-CoV-2 RNAs were detected in all human semen. The ACE2 and TMPRSS2 expression was undetectable in 26 samples, whereas 23 samples only had a detectable TMPRSS2 expression and 4 only had an ACE2 expression. The remaining 3 expressed both ACE2 and TMPRSS2. CONCLUSIONS: Severe acute respiratory syndrome coronavirus 2 could not be found in the semen of a cohort of young to middle-aged Asian men with mild acute SARS-CoV-2 infection. However, there was a detectable expression of ACE2 and TMPRSS2 in semen, although not causal, and it may be correlated to changes in male hormone profiles and male age.

Validation of a multi-gene qPCR-based pharmacogenomics panel across major ethnic groups in Singapore and Indonesia.

Aim: The clinical utility of pharmacogenomics (PGx) has been gaining traction alongside growing evidence that adverse drug reactions (ADRs) have significant genetic associations. Nala PGx Core® is a multi-gene qPCR-based panel of 20 allele variants, comprising 18 SNPs and two CYP2D6 copy number markers across four pharmacogenes - CYP2C9, CYP2C19, CYP2D6 and SLCO1B1. Methods: In this study, we validated the performance of Nala PGx Core® against benchmark methods, on the Singaporean and Indonesian populations. Results & conclusion: Nala PGx Core® demonstrated robust and accurate genotyping when compared with other established benchmarks. Furthermore, the panel successfully characterized alleles of clinical relevance, such as CYP2D6*10 and CYP2D6*36, across major ethnic groups present of Singapore and Indonesia, suggesting its potential for adoption in clinical workflows regionally.

An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing.

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.

Design and Multicenter Clinical Validation of a 3-Dimensionally Printed Nasopharyngeal Swab for SARS-CoV-2 Testing.

Importance: Three-dimensionally printed nasopharyngeal swabs (3DP swabs) have been used to mitigate swab shortages during the coronavirus disease 2019 (COVID-19) pandemic. Clinical validation for diagnostic accuracy and consistency, as well as patient acceptability, is crucial to evaluate the swab's performance. Objective: To determine the accuracy and acceptability of the 3DP swab for identifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Design, Setting, and Participants: A diagnostic study was conducted from May to July 2020 at 2 tertiary care centers in Singapore with different reference swabs (FLOQSwab [COPAN Diagnostics] or Dacron swab [Deltalab]) and swab processing techniques (wet or dry) to evaluate the performance of the 3DP swab compared with traditional, standard-of-care nasopharyngeal swabs used in health care institutions. The participants were patients with COVID-19 in the first 2 weeks of illness and controls with acute respiratory illness with negative test results for SARS-CoV-2. Paired nasopharyngeal swabs were obtained from the same nostril and tested for SARS-CoV-2 by reverse-transcriptase polymerase chain reaction. The sequence of swabs was randomized based on odd and even participant numbers. Main Outcomes and Measures: Primary outcome measures were overall agreement (OA), positive percentage agreement (PPA), and negative percentage agreement of the 3DP swab compared with reference swabs. Secondary outcome measures were the correlation of cycle threshold (Ct) values of both swabs. Results: The mean (SD) age of participants was 45.4 (13.1) years, and most participants were men (87 of 89 [97.8%]), in keeping with the epidemiology of the COVID-19 pandemic in Singapore. A total of 79 patients with COVID-19 and 10 controls were recruited. Among the patients with COVID-19, the overall agreement and PPA of the 3DP swab was 91.1% and 93.5%, respectively, compared with reference swabs. The PPA was 100% for patients with COVID-19 who were tested within the first week of illness. All controls tested negative. The reverse-transcriptase polymerase chain reaction Ct values for the ORF1ab and E-gene targets showed a strong correlation (intraclass correlations coefficient, 0.869-0.920) between the 3DP and reference swab on independent testing at each institution despite differences in sample processing. Discordant results for both gene targets were observed only at high Ct values. Conclusions and Relevance: In this diagnostic study of 79 patients with COVID-19 and 10 controls, the 3DP swab performed accurately and consistently across health care institutions and could help mitigate strained resources in the escalating COVID-19 pandemic.

Clinical Diagnostic Study of a Novel Injection Molded Swab for SARS-Cov-2 Testing.

INTRODUCTION: The gold standard for COVID-19 diagnosis is currently a real-time reverse transcriptase polymerase chain reaction (RT-PCR) to detect SARS-CoV-2. This is most commonly performed on respiratory secretions obtained via a nasopharyngeal swab. Due to supply chain limitations and high demand worldwide because of the COVID-19 pandemic, access to commercial nasopharyngeal swabs has not been assured. 3D printing methods have been used to meet the shortfall. For longer-term considerations, 3D printing may not compare well with injection molding as a production method due to the challenging scalability and greater production costs of 3D printing. METHODS: To secure sufficient nasopharyngeal swab availability for our national healthcare system, we designed a novel injection molded nasopharyngeal swab (the IM2 swab). We performed a clinical diagnostic study comparing the IM2 swab to the Copan FLOQSwab. Forty patients with a known diagnosis of COVID-19 and 10 healthy controls were recruited. Paired nasopharyngeal swabs were obtained from the same nostril of each participant and tested for SARS-CoV-2 by RT-PCR. RESULTS: When compared to the Copan FLOQswab, results from the IM2 swab displayed excellent overall agreement and positive percent agreement of 96.0% and 94.9%, respectively. There was no significant difference in mean RT-PCR cycle threshold values for the ORF1ab (28.05 vs. 28.03, p = 0.97) and E-gene (29.72 vs. 29.37, p = 0.64) targets, respectively. We did not observe any significant adverse events and there was no significant difference in patient-reported pain. CONCLUSION: In summary, the IM2 nasopharyngeal swab is a clinically safe, highly accurate option to commercial nasopharyngeal swabs.

Practical laboratory considerations amidst the COVID-19 outbreak: early experience from Singapore.

The coronavirus disease 2019 (COVID-19) is a zoonotic viral infection originating from Wuhan, China in December 2019. The World Health Organization has classified this pandemic as a global health emergency due to its virulent nature of transmission, which may lead to acute respiratory distress syndrome. Singapore's health ministry has responded with enhanced surveillance of COVID-19 for all suspected pneumonia cases, further increasing the volume of testing via real-time reverse transcription PCR, as well as samples necessitating stringent infectious control. Collectively, this has implications on the total testing process, laboratory operations and its personnel due to biosafety concerns. Turnaround time for routine testing may also be affected. The aim of this article is to present our tertiary institution's early experience with managing this emerging crisis and offer practical considerations for the preanalytical, analytical and postanalytical phases of laboratory testing in this cohort of patients.

Pregnancy Outcomes in COVID-19: A Prospective Cohort Study in Singapore.

INTRODUCTION: Pregnant women are reported to be at increased risk of severe coronavirus disease 2019 (COVID-19) due to underlying immunosuppression during pregnancy. However, the clinical course of COVID-19 in pregnancy and risk of vertical and horizontal transmission remain relatively unknown. We aim to describe and evaluate outcomes in pregnant women with COVID-19 in Singapore. METHODS: Prospective observational study of 16 pregnant patients admitted for COVID-19 to 4 tertiary hospitals in Singapore. Outcomes included severe disease, pregnancy loss, and vertical and horizontal transmission. RESULTS: Of the 16 patients, 37.5%, 43.8% and 18.7% were infected in the first, second and third trimesters, respectively. Two gravidas aged ≥35 years (12.5%) developed severe pneumonia; one patient (body mass index 32.9kg/m2) required transfer to intensive care. The median duration of acute infection was 19 days; one patient remained reverse transcription polymerase chain reaction (RT-PCR) positive >11 weeks from diagnosis. There were no maternal mortalities. Five pregnancies produced term live-births while 2 spontaneous miscarriages occurred at 11 and 23 weeks. RT-PCR of breast milk and maternal and neonatal samples taken at birth were negative; placenta and cord histology showed non-specific inflammation; and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immunoglobulins were elevated in paired maternal and umbilical cord blood (n=5). CONCLUSION: The majority of COVID-19 infected pregnant women had mild disease and only 2 women with risk factors (obesity, older age) had severe infection; this represents a slightly higher incidence than observed in age-matched non-pregnant women. Among the women who delivered, there was no definitive evidence of mother-to-child transmission via breast milk or placenta.

Relationship of JAK2 (V617F) Allelic Burden with Clinico- Haematological Manifestations of Philadelphia-Negative Myeloproliferative Neoplasms.

JAK2 (V617F) allelic burden is the main genetic driver behind and a potential differentiator between individual myeloproliferative neoplasm (MPN) subtypes. This study aimed to explore the relationship between JAK2 (V617F) allelic burden, MPN subtypes and their clinico-haematological manifestations in a Singapore-based cohort. Analysis was performed on a retrospectively collected dataset of 128 patients diagnosed with JAK2 (V617F) positive Philadelphia-negative MPNs between 2016 to 2017 in Singapore. Genomic analysis was conducted on blood samples via DNA extraction and Droplet Digital Polymerase Chain Reaction (ddPCR). The mean age was 62.4 (SD=14.1). 85 out of the 128 (66.4%) patients were male. There was a statistically significant difference in allelic burdens between the different MPN disease subtypes χ2(3) = 9.064, p=0.028, with essential thrombocytosis (ET) patients having the lowest mean JAK2 percentage allelic burden (26.5%). Patients with an allelic burden >50% had higher leukocyte counts (MWU 1016.5, p=0.001), haemoglobin levels (MWU 1287.0, p=0.045), lactate dehydrogenase levels (MWU 611.5, p=0.001), and lower platelet levels (MWU 1164.0, p=0.008). Subgroup analysis revealed none of these correlations was significant in the ET subgroup. The results are largely in concordance with previous research in Asian cohorts demonstrating the association between allelic burden and clinico-haematological manifestations of MPN. However, in the ET subgroup, the JAK2 (V617F) allelic burden do not correlate positively for haematological parameters which is only seen in Asian patients.
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JAK2S523L, a novel gain-of-function mutation in a critical autoregulatory residue in JAK2V617F- MPNs.

The SH2-JH2 linker domain of JAK2 has been implicated in the negative regulation of JAK2 activity. In 2 patients with myeloproliferative neoplasms (MPNs), we identified and characterized the novel JAK2 mutation S523L, which occurs in a key residue in the linker region. In 1 case, acquisition of JAK2S523L was associated with thrombocytosis and bone marrow megakaryocytic hyperplasia, and there were no other somatic alterations in this patient. The second patient with JAK2S523Lmutation presented with increased hematocrit and had concurrent mutations in RUNX1 and BCORL1. Consistent with the genetic and clinical data, expression of JAK2S523L causes interleukin-3-independent growth in Ba/F3 cells transduced with the erythropoietin receptor by constitutively active Jak2/Stat5 signaling.

Four Patients with COVID-19 and Tuberculosis, Singapore, April-May 2020.

Coronavirus disease (COVID-19) and tuberculosis (TB) developed in 4 foreign workers living in dormitories in Singapore during April-May 2020. Clinical manifestations and atypical radiographic features of COVID-19 led to the diagnosis of TB through positive interferon-gamma release assay and culture results. During the COVID-19 pandemic, TB should not be overlooked.

Recommendations to improve the clinical adoption of NGS-based cancer diagnostics in Singapore.

Next-generation sequencing (NGS)-based diagnostics have demonstrated clinical utility in predicting improved survival benefits with targeted treatment in certain cancer types, and positive cost-benefit in several healthcare systems. However, clinical adoption in Singapore remains low despite commercial availability of these diagnostics. This expert opinion review examines the key challenges to the clinical adoption of NGS-based diagnostics in Singapore, provides recommendations on impactful initiatives to improve adoption, and also offers practical guidance on specific cancer types in which NGS-based diagnostics are appropriate for use in Singapore. Limited patient affordability is one major challenge to clinical adoption of NGS-based diagnostics, which could be improved by enabling patient access to more funds for specific cancer types with clear benefits. Expert opinion based on current evidence and clinical experience supports the upfront use of hotspot panels in advanced non-small cell lung cancer (NSCLC), metastatic colorectal cancer, advanced and recurrent ovarian cancer, and acute myeloid leukemia. Comprehensive genomic profiling could be considered for upfront use in select patients with NSCLC and ovarian cancer, or in refractory patients with the four cancer types. Wider adoption of NGS-based diagnostics will improve the delivery of cancer care in Singapore and Asia-Pacific, and thus lead to better patient outcomes.

A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma.

c-MET receptors are activated in cancers through genomic events like tyrosine kinase domain mutations, juxtamembrane splicing mutation and amplified copy numbers, which can be inhibited by c-MET small molecule inhibitors. Here, we discover that the most common polymorphism known to affect MET gene (N375S), involving the semaphorin domain, confers exquisite binding affinity for HER2 and enables METN375S to interact with HER2 in a ligand-independent fashion. The resultant METN375S/HER2 dimer transduces potent proliferative, pro-invasive and pro-metastatic cues through the HER2 signaling axis to drive aggressive squamous cell carcinomas of the head and neck (HNSCC) and lung (LUSC), and is associated with poor prognosis. Accordingly, HER2 blockers, but not c-MET inhibitors, are paradoxically effective at restraining in vivo and in vitro models expressing METN375S. These results establish METN375S as a biologically distinct and clinically actionable molecular subset of SCCs that are uniquely amenable to HER2 blocking therapies.