Ethnicity Is Associated With Differing Presentation and Outcomes of Patients Undergoing Aortic Valve Replacement for Calcific Aortic Stenosis in Aotearoa New Zealand.

AIM: Surgical aortic valve replacement (SAVR) has been the gold standard for treatment of severe symptomatic aortic stenosis (AS) for decades. We examined whether ethnic differences exist in the presentation and outcomes of patients undergoing aortic valve replacement (AVR) for AS in New Zealand. METHODS: Patients of New Zealand European, Maori, and Pacific Island ethnicities undergoing SAVR with or without other procedures in New Zealand public hospitals from 2017 to 2019 were included. Major postoperative outcomes were compared between ethnic groups, with 30-day mortality being the primary outcome. RESULTS: A total of 1,175 patients were included: 1,085 European, 50 Maori, and 40 Pacific. The mean age was 71.1±9.4 years, and men accounted for more than half of all patients (69.9%). Maori (64.7±9.4 years) and Pacific (65.4±10.1 years) patients were younger when undergoing SAVR compared with European patients (71.7±9.2; analysis of variance p<0.001). Maori and Pacific patients had a higher prevalence of diabetes, poorer renal function, and worse left ventricular function; 30-day mortality was higher in Maori and Pacific compared with European patients (6% and 10% vs 2.4%, respectively; Fisher's exact test p=0.011), with odds ratio of 3.06 (95% confidence interval [CI] 0.88-10.66) for Maori patients after adjustment for EuroSCORE II and odds ratio of 5.23 (95% CI 1.79-16.07) for Pacific patients. CONCLUSIONS: There are significant differences in presentation and outcomes of patients undergoing AVR in New Zealand. Maori and Pacific patients undergo SAVR at a younger age, have more preoperative comorbidities, and have higher rates of 30-day mortality than European patients.

Prevalence of Chlamydia trachomatis Infection in Young Women and Associated Predictors.

BACKGROUND: Chlamydia trachomatis (CT) infection remains highly prevalent, and young women are disproportionately affected. Most CT-infected women are asymptomatic, and their infection often goes unrecognized and untreated. We hypothesized that testing for active CT infection with molecular diagnostics and obtaining a reported history of CT infection underestimate the prevalence of current and past CT infection, and incorporating serum CT antibody testing in addition to these other prevalence measures would generate more accurate estimates of the prevalence of CT infection in asymptomatic young women. METHODS: We enrolled 362 asymptomatic women aged 16 to 29 years at 4 different clinical settings in Birmingham, AL, between August 2016 and January 2020 and determined the prevalence of CT infection based on having 1 or more of the following prevalence measures: an active urogenital CT infection based on molecular testing, reported prior CT infection, and/or being CT seropositive. Multivariable regression analysis was used to determine predictors of the prevalence of CT infection after adjustment for participant characteristics. RESULTS: The prevalence of CT infection was 67.7% (95% confidence interval, 62.6%-72.5%). Addition of CT antibody testing to the other individual prevalence measures more than doubled the CT infection prevalence. Non-Hispanic Black race, reported prior gonorrhea, and reported prior trichomoniasis predicted a higher prevalence of CT infection. CONCLUSIONS: More than half of women were unaware of ever having CT infection, suggesting many were at risk for CT-associated reproductive complications. These data reinforce the need to adhere to chlamydia screening guidelines and to increase screening coverage in those at risk.

m6aViewer: software for the detection, analysis, and visualization of N6-methyladenosine peaks from m6A-seq/ME-RIP sequencing data.

Recent methods for transcriptome-wide N6-methyladenosine (m6A) profiling have facilitated investigations into the RNA methylome and established m6A as a dynamic modification that has critical regulatory roles in gene expression and may play a role in human disease. However, bioinformatics resources available for the analysis of m6A sequencing data are still limited. Here, we describe m6aViewer-a cross-platform application for analysis and visualization of m6A peaks from sequencing data. m6aViewer implements a novel m6A peak-calling algorithm that identifies high-confidence methylated residues with more precision than previously described approaches. The application enables data analysis through a graphical user interface, and thus, in contrast to other currently available tools, does not require the user to be skilled in computer programming. m6aViewer and test data can be downloaded here: http://dna2.leeds.ac.uk/m6a.

Mycoplasma genitalium Coinfection in Women With Chlamydia trachomatis Infection.

We evaluated the prevalence of Mycoplasma genitalium coinfection in 302 chlamydia-infected women seen at a sexually transmitted disease clinic in Birmingham, AL. M genitalium coinfection was detected in 22 (7.3%). No participant characteristics predicted coinfection. Among coinfected women, M genitalium was detected again in 6 (28.6%) of 21 women returning for a 3-month follow-up visit after azithromycin treatment.

Exploring the surfaceome of Ewing sarcoma identifies a new and unique therapeutic target.

The cell surface proteome of tumors mediates the interface between the transformed cells and the general microenvironment, including interactions with stromal cells in the tumor niche and immune cells such as T cells. In addition, the cell surface proteome of individual cancers defines biomarkers for that tumor type and potential proteins that can be the target of antibody-mediated therapy. We have used next-generation deep RNA sequencing (RNA-seq) coupled to an in-house database of genes encoding cell surface proteins (herein referred to as the surfaceome) as a tool to define a cell surface proteome of Ewing sarcoma compared with progenitor mesenchymal stem cells. This subtractive RNA-seq analysis revealed a specific surfaceome of Ewing and showed unexpectedly that the leucine-rich repeat and Ig domain protein 1 (LINGO1) is expressed in over 90% of Ewing sarcoma tumors, but not expressed in any other somatic tissue apart from the brain. We found that the LINGO1 protein acts as a gateway protein internalizing into the tumor cells when engaged by antibody and can carry antibody conjugated with drugs to kill Ewing sarcoma cells. Therefore, LINGO1 is a new, unique, and specific biomarker and drug target for the treatment of Ewing sarcoma.

How to analyse the spatiotemporal tumour samples needed to investigate cancer evolution: A case study using paired primary and recurrent glioblastoma.

Many traits of cancer progression (e.g., development of metastases or resistance to therapy) are facilitated by tumour evolution: Darwinian selection of subclones with distinct genotypes or phenotypes that enable such progression. Characterising these subclones provide an opportunity to develop drugs to better target their specific properties but requires the accurate identification of somatic mutations shared across multiple spatiotemporal tumours from the same patient. Current best practices for calling somatic mutations are optimised for single samples, and risk being too conservative to identify shared mutations with low prevalence in some samples. We reasoned that datasets from multiple matched tumours can be used for mutual validation and thus propose an adapted two-stage approach: (1) low-stringency mutation calling to identify mutations shared across samples irrespective of the weight of evidence in a single sample; (2) high-stringency mutation calling to further characterise mutations present in a single sample. We applied our approach to three-independent cohorts of paired primary and recurrent glioblastoma tumours, two of which have previously been analysed using existing approaches, and found that it significantly increased the amount of biologically relevant shared somatic mutations identified. We also found that duplicate removal was detrimental when identifying shared somatic mutations. Our approach is also applicable when multiple datasets e.g. DNA and RNA are available for the same tumour.

Deficiency of the myogenic factor MyoD causes a perinatally lethal fetal akinesia.

BACKGROUND: Lethal fetal akinesia deformation sequence (FADS) describes a clinically and genetically heterogeneous phenotype that includes fetal akinesia, intrauterine growth retardation, arthrogryposis and developmental anomalies. Affected babies die as a result of pulmonary hypoplasia. We aimed to identify the underlying genetic cause of this disorder in a family in which there were three affected individuals from two sibships. METHODS: Autosomal-recessive inheritance was suggested by a family history of consanguinity and by recurrence of the phenotype between the two sibships. We performed exome sequencing of the affected individuals and their unaffected mother, followed by autozygosity mapping and variant filtering to identify the causative gene. RESULTS: Five autozygous regions were identified, spanning 31.7 Mb of genomic sequence and including 211 genes. Using standard variant filtering criteria, we excluded all variants as being the likely pathogenic cause, apart from a single novel nonsense mutation, c.188C>A p.(Ser63*) (NM_002478.4), in MYOD1. This gene encodes an extensively studied transcription factor involved in muscle development, which has nonetheless not hitherto been associated with a hereditary human disease phenotype. CONCLUSIONS: We provide the first description of a human phenotype that appears to result from MYOD1 mutation. The presentation with FADS is consistent with a large body of data demonstrating that in the mouse, MyoD is a major controller of precursor cell commitment to the myogenic differentiation programme.

OVA: integrating molecular and physical phenotype data from multiple biomedical domain ontologies with variant filtering for enhanced variant prioritization.

MOTIVATION: Exome sequencing has become a de facto standard method for Mendelian disease gene discovery in recent years, yet identifying disease-causing mutations among thousands of candidate variants remains a non-trivial task. RESULTS: Here we describe a new variant prioritization tool, OVA (ontology variant analysis), in which user-provided phenotypic information is exploited to infer deeper biological context. OVA combines a knowledge-based approach with a variant-filtering framework. It reduces the number of candidate variants by considering genotype and predicted effect on protein sequence, and scores the remainder on biological relevance to the query phenotype.We take advantage of several ontologies in order to bridge knowledge across multiple biomedical domains and facilitate computational analysis of annotations pertaining to genes, diseases, phenotypes, tissues and pathways. In this way, OVA combines information regarding molecular and physical phenotypes and integrates both human and model organism data to effectively prioritize variants. By assessing performance on both known and novel disease mutations, we show that OVA performs biologically meaningful candidate variant prioritization and can be more accurate than another recently published candidate variant prioritization tool. AVAILABILITY AND IMPLEMENTATION: OVA is freely accessible at http://dna2.leeds.ac.uk:8080/OVA/index.jsp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: umaan@leeds.ac.uk.

Enhanced diagnostic yield in Meckel-Gruber and Joubert syndrome through exome sequencing supplemented with split-read mapping.

BACKGROUND: The widespread adoption of high-throughput sequencing technologies by genetic diagnostic laboratories has enabled significant expansion of their testing portfolios. Rare autosomal recessive conditions have been a particular focus of many new services. Here we report a cohort of 26 patients referred for genetic analysis of Joubert (JBTS) and Meckel-Gruber (MKS) syndromes, two clinically and genetically heterogeneous neurodevelopmental conditions that define a phenotypic spectrum, with MKS at the severe end. METHODS: Exome sequencing was performed for all cases, using Agilent SureSelect v5 reagents and Illumina paired-end sequencing. For two cases medium-coverage (9×) whole genome sequencing was subsequently undertaken. RESULTS: Using a standard analysis pipeline for the detection of single nucleotide and small insertion or deletion variants, molecular diagnoses were confirmed in 12 cases (4%). Seeking to determine whether our cohort harboured pathogenic copy number variants (CNV), in JBTS- or MKS-associated genes, targeted comparative read-depth analysis was performed using FishingCNV. These analyses identified a putative intragenic AHI1 deletion that included three exons spanning at least 3.4 kb and an intergenic MPP4 to TMEM237 deletion that included exons spanning at least 21.5 kb. Whole genome sequencing enabled confirmation of the deletion-containing alleles and precise characterisation of the mutation breakpoints at nucleotide resolution. These data were validated following development of PCR-based assays that could be subsequently used for "cascade" screening and/or prenatal diagnosis. CONCLUSIONS: Our investigations expand the AHI1 and TMEM237 mutation spectrum and highlight the importance of performing CNV screening of disease-associated genes. We demonstrate a robust increasingly cost-effective CNV detection workflow that is applicable to all MKS/JBTS referrals.

Rapid Detection of Rare Deleterious Variants by Next Generation Sequencing with Optional Microarray SNP Genotype Data.

Autozygosity mapping is a powerful technique for the identification of rare, autosomal recessive, disease-causing genes. The ease with which this category of disease gene can be identified has greatly increased through the availability of genome-wide SNP genotyping microarrays and subsequently of exome sequencing. Although these methods have simplified the generation of experimental data, its analysis, particularly when disparate data types must be integrated, remains time consuming. Moreover, the huge volume of sequence variant data generated from next generation sequencing experiments opens up the possibility of using these data instead of microarray genotype data to identify disease loci. To allow these two types of data to be used in an integrated fashion, we have developed AgileVCFMapper, a program that performs both the mapping of disease loci by SNP genotyping and the analysis of potentially deleterious variants using exome sequence variant data, in a single step. This method does not require microarray SNP genotype data, although analysis with a combination of microarray and exome genotype data enables more precise delineation of disease loci, due to superior marker density and distribution.

Robust diagnostic genetic testing using solution capture enrichment and a novel variant-filtering interface.

Targeted hybridization enrichment prior to next-generation sequencing is a widespread method for characterizing sequence variation in a research setting, and is being adopted by diagnostic laboratories. However, the number of variants identified can overwhelm clinical laboratories with strict time constraints, the final interpretation of likely pathogenicity being a particular bottleneck. To address this, we have developed an approach in which, after automatic variant calling on a standard unix pipeline, subsequent variant filtering is performed interactively, using AgileExomeFilter and AgilePindelFilter (http://dna.leeds.ac.uk/agile), tools designed for clinical scientists with standard desktop computers. To demonstrate the method's diagnostic efficacy, we tested 128 patients using (1) a targeted capture of 36 cancer-predisposing genes or (2) whole-exome capture for diagnosis of the genetically heterogeneous disorder primary ciliary dyskinesia (PCD). In the cancer cohort, complete concordance with previous diagnostic data was achieved across 793 variant genotypes. A high yield (42%) was also achieved for exome-based PCD diagnosis, underscoring the scalability of our method. Simple adjustments to the variant filtering parameters further allowed the identification of a homozygous truncating mutation in a presumptive new PCD gene, DNAH8. These tools should allow diagnostic laboratories to expand their testing portfolios flexibly, using a standard set of reagents and techniques.

Detection of somatic mutations in tumors using unaligned clonal sequencing data.

Most cancers arise and evolve as a consequence of somatic mutations. These mutations influence tumor behavior and clinical outcome. Consequently, there is considerable interest in identifying somatic variants within specific genes (such as BRAF, KRAS and EGFR) so that chemotherapy can be tailored to the patient's tumor genotype rather than using a generic treatment based on histological diagnosis alone. Owing to the heterogeneous nature of tumors, a somatic mutation may be present in only a subset of cells, necessitating the use of quantitative techniques to detect rare variants. The highly quantitative nature of next-generation sequencing (NGS), together with the ability to multiplex numerous samples, makes NGS an attractive choice with which to screen for somatic variants. However, the large volumes of sequence data present significant difficulties when applying NGS for the detection of somatic mutations. To alleviate this, we have developed methodologies including a set of data analysis programs, which allow the rapid screening of multiple formalin-fixed, paraffin-embedded samples for the presence of specified somatic variants using unaligned Illumina NGS data.

Toward complete miniaturisation of flow injection analysis systems: microfluidic enhancement of chemiluminescent detection.

Conventional flow injection systems for aquatic environmental analysis typically comprise large laboratory benchscale equipment, which place considerable constraints for portable field use. Here, we demonstrate the use of an integrated acoustically driven microfluidic mixing scheme to enhance detection of a chemiluminescent species tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate-a common chemiluminescent reagent widely used for the analysis of a wide range of compounds such as illicit drugs, pharmaceuticals, and pesticides-such that rapid in-line quantification can be carried out with sufficient on-chip sensitivity. Specifically, we employ surface acoustic waves (SAWs) to drive intense chaotic streaming within a 100 µL chamber cast in polydimethoxylsiloxane (PDMS) atop a microfluidic chip consisting of a single crystal piezoelectric material. By optimizing the power, duration, and orientation of the SAW input, we show that the mixing intensity of the sample and reagent fed into the chamber can be increased by one to two orders of magnitude, leading to a similar enhancement in the detection sensitivity of the chemiluminescent species and thus achieving a theoretical limit of detection of 0.02 ppb (0.2 nM) of l-proline-a decade improvement over the industry gold-standard and two orders of magnitude more sensitive than that achievable with conventional systems-simply using a portable photodetector and without requiring sample preconcentration. This on-chip microfluidic mixing strategy, together with the integrated miniature photodetector and the possibility for chip-scale microfluidic actuation, then alludes to the attractive possibility of a completely miniaturized platform for portable field-use microanalytical systems.

Diagnostic whole genome sequencing and split-read mapping for nucleotide resolution breakpoint identification in CNTNAP2 deficiency syndrome.

Whole genome sequencing (WGS) has the potential to report on all types of genetic abnormality, thus converging diagnostic testing on a single methodology. Although WGS at sufficient depth for robust detection of point mutations is still some way from being affordable for diagnostic purposes, low-coverage WGS is already an excellent method for detecting copy number variants ("CNVseq"). We report on a family in which individuals presented with a presumed autosomal recessive syndrome of severe intellectual disability and epilepsy. Array comparative genomic hybridization (CGH) analysis had revealed a homozygous deletion apparently lying within intron 3 of CNTNAP2. Since this was too small for confirmation by FISH, CNVseq was used, refining the extent of this mutation to approximately 76.8 kb, encompassing CNTNAP2 exon 3 (an out-of-frame deletion). To characterize the precise breakpoints and provide a rapid molecular diagnostic test, we resequenced the CNVseq library at medium coverage and performed split read mapping. This yielded information for a multiplex polymerase chain reaction (PCR) assay, used for cascade screening and/or prenatal diagnosis in this family. This example demonstrates a rapid, low-cost approach to converting molecular cytogenetic findings into robust PCR-based tests.

Simple and efficient identification of rare recessive pathologically important sequence variants from next generation exome sequence data.

Massively parallel ("next generation") DNA sequencing (NGS) has quickly become the method of choice for seeking pathogenic mutations in rare uncharacterized monogenic diseases. Typically, before DNA sequencing, protein-coding regions are enriched from patient genomic DNA, representing either the entire genome ("exome sequencing") or selected mapped candidate loci. Sequence variants, identified as differences between the patient's and the human genome reference sequences, are then filtered according to various quality parameters. Changes are screened against datasets of known polymorphisms, such as dbSNP and the 1000 Genomes Project, in the effort to narrow the list of candidate causative variants. An increasing number of commercial services now offer to both generate and align NGS data to a reference genome. This potentially allows small groups with limited computing infrastructure and informatics skills to utilize this technology. However, the capability to effectively filter and assess sequence variants is still an important bottleneck in the identification of deleterious sequence variants in both research and diagnostic settings. We have developed an approach to this problem comprising a user-friendly suite of programs that can interactively analyze, filter and screen data from enrichment-capture NGS data. These programs ("Agile Suite") are particularly suitable for small-scale gene discovery or for diagnostic analysis.

Rare Case of Cavernous Haemangioma of the Right Atrium with Probable Hepatic Haemangioma.

Cardiac haemangiomas are rare causes of atrial masses. This case report is of a 44-year-old male who presented with a right atrial mass that was found incidentally on a CT performed for renal colic. The mass was further investigated with a transthoracic echocardiogram that showed that it was echodense and arising from the Eustachian valve in the right atrium. Coronary angiogram revealed large well-developed atrial branches that crossed superiorly over the left atrium and entered the mass in the right atrium. Surgical resection was undertaken, and this confirmed that the mass had a fleshy, encapsulated appearance with a sessile stalk. Histology demonstrated a cavernous haemangioma. The patient had a residual small defect in the interatrial septum postoperatively but otherwise made a good recovery.

Patient expectations of cosmetic appearance after complex facial surgery in a low-income country.

Managing aesthetic expectations for patients post facial operations can be challenging in the high-income countries, yet alone in low-income countries. This cohort study involved patients undergoing facial operations during the October 2018 Facing Africa charitable surgical trip at the Nordic Medical Centre in Addis Ababa, Ethiopia. Twenty-one patients were shown pre and postoperative photographs of patients who had operations for similar facial pathologies on a prior surgical mission. They were then interviewed after their own operations also in regards to their own appearance. Admiration (n = 8, 38.1%), followed by trust (n = 6, 28.6%) were the most common emotions expressed by patients after seeing the pre and postoperative clinical photographs. Joy (n = 9, 42.9%) and admiration (n = 9, 42.9%) were most commonly felt by the patients after seeing their own appearance postoperatively. Utilisation of pre and postoperative photographs of patients who underwent similar procedures should help prospective patients prepare for often what is quite a dramatic change to their appearance.

Clinical quality indicators of pathways to oncological lung surgery.

AIMS: Multidisciplinary team (MDT) meetings are a standard of care for lung cancer management in many regions around the world. Clinical quality indicators (CQIs) can be used to assess the proficiency of these multidisciplinary teams and compare their performance against those recommended by local and international guidelines. The effectiveness of the lung cancer MDT meeting at Dunedin Public Hospital has been evaluated using CQIs with a focus on the timeliness of surgical management. METHODS: Medical records for all 108 patients who underwent curative intent oncological lung surgery at Dunedin Public Hospital between 2014-2020 were obtained. All patients were discussed at the lung cancer MDT meeting. Performance in six CQIs were evaluated as per the results below. RESULTS: The CQI for timing of referral to first contact with a respiratory medicine specialist was met in all years studied by mean days. In all years bars for 2014 and 2017, the standard for time by mean days from referral date to surgery was met. In 2017, the mean time to surgery exceeded this standard by only one day. The mean time between respiratory specialist review and surgery was less than 56 days in all years except for 2014. By mean days, 2018 was the only year that surgery was performed within 31 days of discussion at the lung cancer MDT. Computed tomography (CT) guided biopsies and endobronchial ultrasound (EBUS) were only performed within a mean of seven days in only two years (2015 and 2017) out of the seven years of data. The target of all patients with curative small or non-small cell lung cancer receiving a positron emission tomography (PET) scan was not achieved in any year. Post-operative upstaging was more frequent than downstaging (19.4% vs 14.8%), and 71.4% of those upstaged received a PET scan pre-operatively. Maori patients and those with significant socio-economic deprivation were less likely to meet standards of lung cancer care. CONCLUSIONS: Between 2014-2020, the standards for lung cancer management in surgical patients were frequently achieved as measured by mean days. However, a target of ≥95% (90% for CQI 2; 100% for CQI 6) of patients receiving care at the standard was rarely met. Timing of CT biopsy and EBUS was consistently longer than recommended, and pre-operative PET utilisation was less than 100%. Thus, there is still room for improvement in surgical lung cancer management in the Southern Health District.

Kaposi's sarcoma-associated herpesvirus RTA promotes degradation of the Hey1 repressor protein through the ubiquitin proteasome pathway.

The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional repressor proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional repressor protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the corepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) Rta and cellular HMGB1 proteins synergistically transactivate the KSHV ORF50 promoter.

Kaposi's sarcoma-associated herpesvirus 'replication transcriptional activator' (Rta) plays a critical role in the switch from latency to lytic replication. Rta upregulates several lytic KSHV genes, including its own, through multiple mechanisms. We demonstrate that cellular HMGB1 binds and synergistically upregulates the ORF50 promoter in conjunction with Rta. No direct interaction between Rta and HMGB1 was observed, however a ternary complex is formed in the presence of Oct1. Furthermore, deletion of an Oct-1 binding site within the ORF50 promoter ablates the HMGB1-mediated synergistic response. These results suggest Rta autostimulation may be mediated by a transient complex involving Oct1 and HMGB1.