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Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1. Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production., (© 2023. The Author(s).)

Asian populations are under-represented in human genomics research. Here, we characterize clinically significant genetic variation in 9051 genomes representing East Asian, South Asian, and severely under-represented Austronesian-speaking Southeast Asian ancestries. We observe disparate genetic risk burden attributable to ancestry-specific recurrent variants and identify individuals with variants specific to ancestries discordant to their self-reported ethnicity, mostly due to cryptic admixture. About 27% of severe recessive disorder genes with appreciable carrier frequencies in Asians are missed by carrier screening panels, and we estimate 0.5% Asian couples at-risk of having an affected child. Prevalence of medically-actionable variant carriers is 3.4% and a further 1.6% harbour variants with potential for pathogenic classification upon additional clinical/experimental evidence. We profile 23 pharmacogenes with high-confidence gene-drug associations and find 22.4% of Asians at-risk of Centers for Disease Control and Prevention Tier 1 genetic conditions concurrently harbour pharmacogenetic variants with actionable phenotypes, highlighting the benefits of pre-emptive pharmacogenomics. Our findings illuminate the diversity in genetic disease epidemiology and opportunities for precision medicine for a large, diverse Asian population., (© 2022. The Author(s).)

Most aldosterone-producing adenomas (APAs) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell clusters of normal adrenal gland suggests a requirement for codriver mutations in APAs. Here we identified gain-of-function mutations in both CTNNB1 and GNA11 by whole-exome sequencing of 3/41 APAs. Further sequencing of known CTNNB1-mutant APAs led to a total of 16 of 27 (59%) with a somatic p.Gln209His, p.Gln209Pro or p.Gln209Leu mutation of GNA11 or GNAQ. Solitary GNA11 mutations were found in hyperplastic zona glomerulosa adjacent to double-mutant APAs. Nine of ten patients in our UK/Irish cohort presented in puberty, pregnancy or menopause. Among multiple transcripts upregulated more than tenfold in double-mutant APAs was LHCGR, the receptor for luteinizing or pregnancy hormone (human chorionic gonadotropin). Transfections of adrenocortical cells demonstrated additive effects of GNA11 and CTNNB1 mutations on aldosterone secretion and expression of genes upregulated in double-mutant APAs. In adrenal cortex, GNA11/Q mutations appear clinically silent without a codriver mutation of CTNNB1., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Family history has traditionally been an essential part of clinical care to assess health risks. However, declining sequencing costs have precipitated a shift towards genomics-first approaches in population screening programs rendering the value of family history unknown. We evaluated the utility of incorporating family history information for genomic sequencing selection., Methods: To ascertain the relationship between family histories on such population-level initiatives, we analysed whole genome sequences of 1750 research participants with no known pre-existing conditions, of which half received comprehensive family history assessment of up to four generations, focusing on 95 cancer genes., Results: Amongst the 1750 participants, 866 (49.5%) had high-quality standardised family history available. Within this group, 73 (8.4%) participants had an increased family history risk of cancer (increased FH risk cohort) and 1 in 7 participants (n = 10/73) carried a clinically actionable variant inferring a sixfold increase compared with 1 in 47 participants (n = 17/793) assessed at average family history cancer risk (average FH risk cohort) (p = 0.00001) and a sevenfold increase compared to 1 in 52 participants (n = 17/884) where family history was not available (FH not available cohort) (p = 0.00001). The enrichment was further pronounced (up to 18-fold) when assessing only the 25 cancer genes in the American College of Medical Genetics (ACMG) Secondary Findings (SF) genes. Furthermore, 63 (7.3%) participants had an increased family history cancer risk in the absence of an apparent clinically actionable variant., Conclusions: These findings demonstrate that the collection and analysis of comprehensive family history and genomic data are complementary and in combination can prioritise individuals for genomic analysis. Thus, family history remains a critical component of health risk assessment, providing important actionable data when implementing genomics screening programs., Trial Registration: ClinicalTrials.gov NCT02791152 . Retrospectively registered on May 31, 2016.

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Morphological awareness, the ability to manipulate the smallest units of meaning, is critical for Chinese literacy. This is because Chinese characters typically reflect the morphemic, or morpho-syllabic units of language. Yet, the neurocognitive mechanisms underlying Chinese speakers' morphological processing remain understudied. Proficient readers (N = 14) completed morphological and phonological judgment tasks in Chinese, in both auditory and visual modalities, during fMRI imaging. Key to our inquiry were patterns of activation in left temporal regions, especially the superior temporal gyrus, which is critical for phonological processing and reading success. The findings revealed that morphological tasks elicited robust activation in superior and middle temporal regions commonly associated with automated phonological and lexico-semantic analyses. In contrast, the rhyme judgment task elicited greater activation in left frontal lobe regions, reflecting the analytical complexity of sound-to-print mapping in Chinese. The findings suggest that left temporal regions are sensitive to salient morpho-syllabic characteristics of a given language., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: CaM (calmodulin) mutations are associated with congenital arrhythmia susceptibility (calmodulinopathy) and are most often de novo. In this report, we sought to broaden the genotype-phenotype spectrum of calmodulinopathies with 2 novel calmodulin mutations and to investigate mosaicism in 2 affected families., Methods: CaM mutations were identified in 4 independent cases by DNA sequencing. Biochemical and electrophysiological studies were performed to determine functional consequences of each mutation., Results: Genetic studies identified 2 novel CaM variants ( CALM3 -E141K in 2 cases; CALM1 -E141V) and one previously reported CaM pathogenic variant ( CALM3 -D130G) among 4 probands with shared clinical features of prolonged QTc interval (range 505-725 ms) and documented ventricular arrhythmia. A fatal outcome occurred for 2 of the cases. The parents of all probands were asymptomatic with normal QTc duration. However, 2 of the families had multiple affected offspring or multiple occurrences of intrauterine fetal demise. The mother from the family with recurrent intrauterine fetal demise exhibited the CALM3 -E141K mutant allele in 25% of next-generation sequencing reads indicating somatic mosaicism, whereas CALM3 -D130G was present in 6% of captured molecules of the paternal DNA sample, also indicating mosaicism. Two novel mutations (E141K and E141V) impaired Ca 2+ binding affinity to the C-domain of CaM. Human-induced pluripotent stem cell-derived cardiomyocytes overexpressing mutant or wild-type CaM showed that both mutants impaired Ca 2+ -dependent inactivation of L-type Ca 2+ channels and prolonged action potential duration., Conclusions: We report 2 families with somatic mosaicism associated with arrhythmogenic calmodulinopathy, and demonstrate dysregulation of L-type Ca 2+ channels by 2 novel CaM mutations affecting the same residue. Parental mosaicism should be suspected in families with unexplained fetal arrhythmia or fetal demise combined with a documented CaM mutation.

Immunodeficiency secondary to anti-interferon-gamma (anti-IFN-γ) autoantibodies was first described in 2004 as an acquired defect in the IFN-γ pathway leading to susceptibility to multiple opportunistic infections, including dimorphic fungi, parasites, and bacteria, especially tuberculosis and non-tuberculous mycobacterium (NTM) species. It has so far only been described in adult patients. We present 2 cases of disseminated NTM infections in otherwise immunocompetent children. A 16-year-old girl with Sweet's syndrome-like neutrophilic dermatosis developed recurrent fever and cervical lymphadenitis secondary to Mycobacterium abscessus. A 10-year-old boy with a history of prolonged fever, aseptic meningitis, aortitis, and arteritis in multiple blood vessels developed thoracic vertebral osteomyelitis secondary to Mycobacterium avium complex. Both patients were found to have positive serum neutralizing anti-IFNγ autoantibodies. Testing for anti-IFNγ autoantibodies should be considered in otherwise healthy immunocompetent hosts with recurrent or disseminated NTM infection. This represents a phenocopy of primary immunodeficiency which has been recently described only in adults. We report the first two cases of this phenomenon to affect children.

Purpose: Genomic studies have demonstrated the necessity of ethnicity-specific population data to ascertain variant pathogenicity for disease diagnosis and treatment. This study examined the carrier prevalence of treatable inherited disorders (TIDs), where early diagnosis of at-risk offspring can significantly improve clinical outcomes., Methods: Existing exome/ genome sequencing data of 831 Singaporeans were aggregated and examined for disease causing variants in 104 genes associated with 80 TIDs., Results: Among the 831 Singaporean participants, genomic variant filtering and analysis identified 1 in 18 individuals (6%) to be carriers amongst one of 13 TIDs. Citrin deficiency and Wilson disease had the highest carrier frequency of 1 in 41, and 1 in 103 individuals, respectively. The pathogenic variants associated with citrin deficiency were 24 times more prevalent in our local cohorts when compared to Western cohorts., Conclusion: This study demonstrates the value of a population specific genomic database to determine true disease prevalence and has enabled the discovery of carrier frequencies of treatable genetic conditions specific to South East Asian populations, which are currently underestimated in existing data sources. This study framework can be adapted to other population groups and expanded to multiple genetic conditions to inform health policies directing precision medicine.

At the time of publication the author Jyn Ling Kuan did not have a master's degree; this has now been amended to BSc. This has now been corrected in the PDF and HTML versions of the article.

We have previously characterised the histone lysine methyltransferase properties of PRDM9, a member of the PRDM family of putative transcriptional regulators. PRDM9 displays broad substrate recognition and methylates a range of histone substrates, including octamers, core histone proteins, and peptides. In the present study, we show that PRDM9 performs intramolecular automethylation on multiple lysine residues localised to a lysine-rich region on the post-SET (suppressor of variegation 3-9, enhancer of zeste and trithorax) domain. PRDM9 automethylation is abolished by a single active-site mutation, C321P, also known to disrupt interactions with S -adenosylmethionine. We have taken an initial step towards tool compound generation through rational design of a substrate-mimic, peptidic inhibitor of PRDM9 automethylation. The discovery of automethylation in PRDM9 adds a new dimension to our understanding of PRDM9 enzymology., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.

Background: In Western cohorts, the prevalence of incidental findings (IFs) or incidentalome, referring to variants in genes that are unrelated to the patient's primary condition, is between 0.86% and 8.8%. However, data on prevalence and type of IFs in Asian population is lacking., Methods: In 2 cohorts of individuals with genomic sequencing performed in Singapore (total n = 377), we extracted and annotated variants in the 56 ACMG-recommended genes and filtered these variants based on the level of pathogenicity. We then analyzed the precise distribution of IFs, class of genes, related medical conditions, and potential clinical impact., Results: We found a total of 41,607 variants in the 56 genes in our cohort of 377 individuals. After filtering for rare and coding variants, we identified 14 potential variants. After reviewing primary literature, only 4 out of the 14 variants were classified to be pathogenic, while an additional two variants were classified as likely pathogenic. Overall, the cumulative prevalence of IFs (pathogenic and likely pathogenic variants) in our cohort was 1.6%., Conclusion: The cumulative prevalence of IFs through genomic sequencing is low and the incidentalome may not be a significant barrier to implementation of genomics for personalized medicine.

The mosquito-borne West Nile virus (WNV) causes a wide range of symptoms ranging from fever to the often fatal viral encephalitis. To date, no vaccine or drug therapy is available. The trypsin-like WNV NS2B-NS3 protease is deemed a plausible drug target and was shown to be inhibited by bovine pancreatic trypsin inhibitor (BPTI), a 58-residue protein isolated from bovine lung. Herein, we report a protein truncation study that resulted in a novel 14-residue cyclic peptide with equipotent inhibitory activity to native BPTI. We believe our truncation strategy can be further applied in the development of peptide-based inhibitors targeting trypsin-like proteases.

Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.

The dengue virus is a mosquito-borne pathogen responsible for an estimated 50-100 million human dengue infections annually. There are currently no approved drugs against this disease, resulting in a major unmet clinical need. The dengue viral NS2B-NS3 protease has been identified as a plausible drug target due to its involvement in viral replication in mammalian host cells. In the past decade, at least 20 dengue NS2B-NS3 protease inhibitors have been reported in the literature with a range of inhibitory activities in protease assays. However, such assays do not shed light on an inhibitor's ability to penetrate human cell membranes where the viral protease resides. In this study, we investigated the antiviral activities of 15 small-molecule and peptide-based NS2B-NS3 inhibitors on dengue serotype 2-infected HuH-7 human hepatocarcinoma cells. Experimental results revealed anthraquinone ARDP0006 (compound 5) to be the most potent inhibitor which reduced dengue viral titer by more than 1 log PFU/mL at 1 μM in our cell-based assays involving HuH-7 and K562 cell lines, suggesting that its scaffold could serve as a lead for further medicinal chemistry studies. Compound 5 was also found to be non-cytotoxic at 1 μM over 3 days incubation on HuH-7 cells using the Alamar Blue cellular toxicity assay., (Copyright © 2015 Elsevier B.V. All rights reserved.)

PRDM proteins have emerged as important regulators of disease and developmental processes. To gain insight into the mechanistic actions of the PRDM family, we have performed comprehensive characterization of a prototype member protein, the histone methyltransferase PRDM9, using biochemical, biophysical and chemical biology techniques. In the present paper we report the first known molecular characterization of a PRDM9-methylated recombinant histone octamer and the identification of new histone substrates for the enzyme. A single C321P mutant of the PR/SET domain was demonstrated to significantly weaken PRDM9 activity. Additionally, we have optimized a robust biochemical assay amenable to high-throughput screening to facilitate the generation of small-molecule chemical probes for this protein family. The present study has provided valuable insight into the enzymology of an intrinsically active PRDM protein.

The Murray Valley encephalitis virus (MVEV) and the West Nile virus (WNV) are mosquito-borne single-stranded RNA Flaviviruses responsible for many cases of viral encephalitis and deaths worldwide. The former is endemic in north Australia and Papua New Guinea while the latter has spread to different parts of the world and was responsible for a recent North American outbreak in 2012, resulting in 243 fatalities. There is currently no approved vaccines or drugs against MVEV and WNV viral infections. A plausible drug target is the viral non-structural NS2B/NS3 protease due to its role in viral replication. This trypsin-like serine protease recognizes and cleaves viral polyproteins at the C-terminal end of an arginine residue, opening an avenue for the development of peptide-based antivirals. This communication compares the P2 and P3 residue preferences of the MVEV and WNV NS2B/NS3 proteases using a series of C-terminal agmatine dipeptides. Our results revealed that both viral enzymes were highly specific toward lysines at the P2 and P3 positions, suggesting that a peptidomimetic viral protease inhibitor developed against one virus should also be active against the other., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker ("linked protease"), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a (1)H-(15)N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV.