Causative Variants for Inherited Cardiac Conditions in a Southeast Asian Population Cohort.

BACKGROUND: Variable penetrance and late-onset phenotypes are key challenges for classifying causal as well as incidental findings in inherited cardiac conditions. Allele frequencies of variants in ancestry-specific populations, along with clinical variant analysis and interpretation, are critical to determine their true significance. METHODS: Here, we carefully reviewed and classified variants in genes associated with inherited cardiac conditions based on a population whole-genome sequencing cohort of 4810 Singaporeans representing Southeast Asian ancestries. RESULTS: Eighty-nine (1.85%) individuals carried either pathogenic or likely pathogenic variants across 25 genes. Forty-six (51.7%) had variants in causal genes for familial hyperlipidemia, but there were also recurrent variants in SCN5A and MYBPC3, causal genes for inherited arrhythmia and cardiomyopathy, which, despite previous reports, we determined to lack criteria for pathogenicity. CONCLUSIONS: Our findings highlight the incidence of disease-related variants in inherited cardiac conditions and emphasize the value of large-scale sequencing in specific ancestries. Follow-up detailed phenotyping and analysis of pedigrees are crucial because assigning pathogenicity will significantly affect clinical management for individuals and their family members.

Adjuvant treatment in cervical, vaginal and vulvar cancer.

Primary surgical management is successful as the sole therapeutic modality in the majority of women with early-stage cervical, vaginal and vulvar cancer, but the presence of certain risk factors in the surgico-pathological specimen indicates a poorer prognosis. Adjuvant treatment can improve overall survival in such cases. Important risk factors in cervical cancer include intermediate-risk factors (large tumor size, deep cervical stromal invasion, lymph-vascular space invasion) and high-risk factors (positive or close margins, lymph nodes, or parametrial involvement). In vulvar cancer, positive margins and lymph nodes are the two most important factors for adjuvant therapy. Radiation therapy has been the mainstay of adjuvant therapy in these cancers, supplemented by chemotherapy. Recent advances have witnessed the inclusion of newer therapeutic modalities such as immunotherapy. This review addresses the current status of various adjuvant therapeutic modalities for these gynecological cancers.

The orphan nuclear receptor NR0B2 could be a novel susceptibility locus associated with microsatellite-stable, APC mutation-negative early-onset colorectal carcinomas with metabolic manifestation.

Colorectal cancer (CRC) is a high incidence cancer and major cause of cancer mortality. Though disease-causing tumor suppressors for major syndromes are well characterized, about 10% of CRC is familial but without mutations in known tumor suppressors. We exhaustively screened 100 polyposis families for APC germline mutations and identified 13, which are APC mutation-negative, microsatellite-stable (MSS), and with undetectable mutation in known tumor suppressors. Whole exome sequencing in three probands uncovered two with germline frameshift NR0B2 mutations, c.293_301delTTGGGTTGGinsAC and c.227delT. Sanger Sequencing identified a third proband with NR0B2 c.157_166delCATCGCACCT frameshift mutation. All three mutations deleted the C-terminus activation/repression domain of NR0B2, thus are loss-of-function mutations. Real-time RT-PCR performed on tumor and matched mucosa of one patient revealed that NR0B2 downstream targets, SMAD3 was derepressed while GLI1 was downregulated in the colonic mucosa compared to healthy controls. Truncated NR0B2 molecule was predicted to have weakened binding with interacting partners SMAD3, GLI1, BCL2, and RXRα, implying perturbation of TGF-ß, Hedgehog, anti-apoptotic and nuclear hormone receptor signaling pathways. Immunostaining also revealed nuclear retention of the most severely truncated NR0B2 molecule compared to the wildtype. Microsatellite and sequencing analysis did not detect loss of wildtype allele in probands' tumors. The patient who acquired somatic KRAS mutation progressed rapidly whist the other two patients manifested with late-onset obesity and diabetes. We propose that haploinsufficiency of NR0B2 is associated with a novel CRC syndrome with metabolic phenotypes.

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.

Procurement, storage and utilization trends of eye banks in India.

Purpose: To study the trends in collection, storage and utilization of donor corneas in eye banks in India. Methods: The data was collected from 12 eye banks in India that collected more than 1000 corneas per year. The retrospective analysis of the parameters like characteristics of the donor and the host, storage media used, number of eyes collected, number of eyes utilized, causes of non-utilization of the tissue and the procedures performed was done. Results: A total of 20,564 eyes were collected by the 12 eye banks during the year 2013-2014. Voluntary eye donation (VED), and hospital cornea retrieval program (HCRP) contributed to 59.6% and 40.4% of tissue procurement respectively. Whole globe enucleation (52.3%) was more commonly performed as compared to in-situ excision of the donor corneas. The most commonly used storage media at all eye banks was McCarey-Kaufman (MK) media (83.3%). The utilization rate of the donor eyes was 50.5%. The most frequent indication for corneal transplantation was infection (active infection - 33.13%, healed infection - 10.78%) followed by Pseudophakic bullous keratopathy (PBK) (13.57%). Full thickness keratoplasty (optical penetrating keratoplasty - 47.23%, therapeutic penetrating keratoplasty - 31.74%) was performed most often followed by endothelial keratoplasty (12.41%) in the developing country. Conclusion: VED still contributes to majority of the donor tissue retrieval in India. The majority of the eye banks still utilize whole globe enucleation technique and store tissues in MK media. Trends from previous years showed a change towards HCRP, in-situ excision technique and preservation in the long-term storage media.

Mutational spectrum of dystrophinopathies in Singapore: Insights for genetic diagnosis and precision therapy.

Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive disorders caused by mutations in the DMD gene. Emerging therapies targeting patients with specific mutations are now becoming a reality for many of these patients. Precise molecular diagnosis is essential to facilitate the identification of possible new treatments for patients in the local context. In this study, we screened 145 dystrophinopathic patients in Singapore and assessed their molecular status for eligibility to current emerging genetic therapies. Overall, 140 (96.5%) of all patients harbored pathogenic DMD mutations comprising 95 exonic deletions (65.5%), 14 exonic duplications (9.7%), and 31 pathogenic small mutations (21.4%). Nonsense and frameshift mutations constitute 83.9% of all the small mutations. We found 71% (103/145) of all Singaporean dystrophinopathy patients to be theoretically amenable for exon skipping, either through skipping of single (53.1%) or multiple exons (17.9%). This approach is applicable to 81.1% (77/95) of patients carrying deletions and 83.9% (26/31) of those with small mutations. Eteplirsen induced skipping of exon 51 is applicable to 12.4% of local patients. Nonsense read-through therapy was found to be applicable in another 12.4% of all patients. Mutation screening is crucial for providing insights into the underlying genetic signature of the disease in the local population and contributes toward existing information on DMD mutations in Asia and globally. This will guide future targeted drug development and clinical trial planning for this disease.

Specific phenotype semantics facilitate gene prioritization in clinical exome sequencing.

Selection and prioritization of phenotype-centric variants remains a challenging part of variant analysis and interpretation in clinical exome sequencing. Phenotype-driven shortlisting of patient-specific gene lists can avoid missed diagnosis. Here, we analyzed the relevance of using primary Human Phenotype Ontology identifiers (HPO IDs) in prioritizing Mendelian disease genes across 30 in-house, 10 previously reported, and 10 recently published cases using three popular web-based gene prioritization tools (OMIMExplorer, VarElect & Phenolyzer). We assessed partial HPO-based gene prioritization using randomly chosen and top 10%, 30%, and 50% HPO IDs based on information content and found high variance within rank ratios across the former vs the latter. This signified that randomly selected less-specific HPO IDs for a given disease phenotype performed poorly by ranking probe gene farther away from the top rank. In contrast, the use of top 10%, 30%, and 50% HPO IDs individually could rank the probe gene among the top 1% in the ranked list of genes that was equivalent to the results when the full list of HPO IDs were used. Hence, we conclude that use of just the top 10% of HPO IDs chosen based on information content is sufficient for ranking the probe gene at top position. Our findings provide practical guidance for utilizing structured phenotype semantics and web-based gene-ranking tools to aid in identifying known as well unknown candidate gene associations in Mendelian disorders.

Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling.

Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA) assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59) while only 42.4% (25/59) of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9%) of tumors screened. There were 3 cases (5.1%) in which no mutations could be detected and germline mutations were detected in 19.5% (8/41) of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59) of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and asymptomatic family members carrying low-penetrance, germline mosaicism or heritable unilateral mutational phenotypes.

Investigating the role of co-substrate-substrate ratio and filter media on the performance of anammox hybrid reactor treating nitrogen rich wastewater.

This study explored the feasibility of using the anammox hybrid reactor (AHR), which combines suspended and attached growth media, for the biodegradation of ammonical nitrogen in wastewater. The study was performed in four laboratory-scale AHRs, inoculated with mixed seed culture (1:1). The anammox process was established by feeding the AHR with synthetic wastewater, containing NH(4)-N and NO(2)-N (1:1), at hydraulic retention time (HRT) of 1 day. The reactors were gradually acclimated to a higher ammonium concentration (1200 mg/l) until the pseudo-steady state was attained. Subsequently, the reactors were operated at various HRTs (0.25-3.0 days) to optimize the HRT and nitrogen loading rate (NLR). The study demonstrated that HRT of 1 day, corresponding to 95.1% of nitrogen removal was optimal. Pearson correlation analysis indicated the strong and positive correlation of HRT and sludge retention time (SRT), whereas the NLR and biomass yield correlated negatively with the nitrogen removal efficiency (NRE). The mass balance of nitrogen showed that a major fraction (79.1%) of the input nitrogen was converted into N2 gas, and 11.25% was utilized in synthesizing the biomass. The filter media in the AHR contributed to an additional 15.4% of ammonium removal and a reduction of 29% in the sludge washout rate. The nitrogen removal kinetics in the AHR followed the modified Stover-Kincannon model, whereas the Lawrence-McCarty model best described the bacterial growth kinetics. The study concludes that the hybrid configuration of the reactor demonstrated promising results and could be suitably applied for industrial applications.

Performance evaluation of the anammox hybrid reactor seeded with mixed inoculum sludge.

Long startup and poor granulation are the major bottlenecks in field-scale application of the anammox (ANaerobic AMMonium OXidation) process. In the present study, the anammox process was investigated in a modified anammox hybrid reactor (AHR) inoculated with mixed seed culture (anoxic and activated sludge). The startup study delineated four distinct phases, i.e. cell lysis, lag phase, activity elevation and stationary phase. Use of mixed seed culture at influent [Formula: see text] ratio (1:1) and hydraulic retention time (HRT) of 1 d led to early startup of the anammox process. The removal efficiencies of [Formula: see text] and [Formula: see text] during acclimation were found to be 94.3% and 96.4%, respectively, at nitrogen loading rate (NLR) of 0.35 kg N/m(3) d. Pearson correlation analysis dictated strong and positive correlation of HRT and sludge retention time (SRT) with nitrogen removal efficiency (NRE) while NLR and sludge loading rate (SLR) were negatively correlated. Attached growth system (AGS) in AHR contributed an additional 11% ammonium removal and reduced the sludge washout rate by 29%. Mass balance of nitrogen revealed that the major fraction (74.1%) of input nitrogen was converted into N2 gas indicating higher substrate conversion efficiency of anammox biomass. Scanning electron microscope (SEM) study of biomass indicated the presence of heterogeneous population of cocci and rod-shaped bacteria of average diameter varying from 1.2 to 1.5 mm. Owing to the features of early start-up, ability to retain high biomass and consistently higher NRE, hybrid reactor configuration seeded with mixed culture offers noble strategy for cultivation of well-compacted anammox granules for field-scale installation.

Human papillomavirus status and gene expression profiles of oropharyngeal and oral cancers from European American and African American patients.

BACKGROUND: Disparities in prevalence, human papillomavirus (HPV) status, and mortality rates for head and neck cancer have been described between African American and European American patients. METHODS: We studied the HPV status and gene expression profiles in 56 oropharyngeal/oral cavity tumors and 9 normal tissue samples from European American and African American patients treated in South Carolina between 2010 and 2012. RESULTS: Overall, 59% of tumors were HPV DNA-positive, but only 48% of those expressed E7 mRNA (HPV-active). The prevalence of HPV-active tumors was 10% in African American patients and 39% in European American patients. Tumors positive for HPV DNA but negative for HPV mRNA exhibited gene expression profiles distinct from those of both HPV-active and HPV-negative cancers, suggesting that HPV DNA-positive/RNA-negative tumors may constitute a unique group. CONCLUSION: This study provides a direct assessment of differential expression patterns in HPV-related oropharyngeal cancer arising from African American and European American patients, for which there is a paucity of data. © 2015 Wiley Periodicals, Inc. Head Neck 00: 000-000, 2015.

A novel strategy for simultaneous removal of nitrogen and organic matter using anaerobic granular sludge in anammox hybrid reactor.

The coexistence of organic matter (OM) and nitrogen in industrial effluent is the major bottleneck in field-scale application of anammox process. The present study emphasized on investigating the role of seeding anaerobic granular sludge towards simultaneous removal of ammonium and OM in anammox hybrid reactor (AHR). The study delineated simultaneous reduction of both OM (94.8%) and nitrogen (96.8%) at optimal COD/N ratio (0.54). Pearson correlation matrix showed positive and strong correlation of ARE (ammonium removal efficiency) and CRE (COD removal efficiency) with NRE (nitrogen removal efficiency). The negative correlation of OLR and COD/TN ratio with NRE indicated that increase in organic loadings may suppress anammox activity. The process inhibition was evaluated using Haldane model considering free ammonia, OM and nitrite as inhibitors. The strategy of using anaerobic granular sludge not only augmented endurance of bacterial communities against OM inhibition but also facilitated simultaneous removal of OM and nitrogen.

A new mathematical model for nitrogen gas production with special emphasis on the role of attached growth media in anammox hybrid reactor.

The present study emphasised on the development of new mathematical models based on mass balance and stoichiometry of nitrogen removal in anammox hybrid reactor (AHR). The performance of AHR at varying hydraulic retention times (HRTs) and nitrogen loading rates (NLRs) revealed that nitrogen removal efficiency (NRE) increases with increase in HRT and was found optimal (89 %) at HRT of 2 days. Mass balance of nitrogen revealed that major fraction (74.1 %) of input nitrogen is converted into N2 gas followed by 11.2 % utilised in biomass synthesis. Attached growth media (AGM) in AHR contributed to an additional 15.4 % ammonium removal and reduced the sludge washout rate by 29 %. This also enhanced the sludge retention capacity of AHR and thus minimised the formation of nitrate in the treated effluent, which is one of the bottlenecks of anammox process. Process kinetics was also studied using various mathematical models. The mass balance model derived from total nitrogen was found most precise and predicted N2 gas with least error (1.68 ± 4.44 %). Model validation for substrate removal kinetics dictated comparatively higher correlation for Grau second-order model (0.952) than modified Stover-Kincannon model (0.920). The study concluded that owing to features of high biomass retention, less nitrate formation and consistently higher nitrogen removal efficiency, this reactor configuration is techno-economically most efficient and viable. The study opens the door for researchers and scientists for pilot-scale testing of AHR leading to its wide industrial application.

The translational significance of epithelial-mesenchymal transition in head and neck cancer.

Positive markers of epithelial-mesenchymal transition (EMT) in head and neck cancers complicate clinical management and are associated with reduced survival. We discuss recent translational discoveries in EMT and suggest additional actionable molecular pathways, biomarkers, and clinical agents.