Next-generation sequencing-based molecular diagnosis of 82 retinitis pigmentosa probands from Northern Ireland

Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by progressive photoreceptor degeneration. An accurate molecular diagnosis is essential for disease characterization and clinical prognoses. A retinal capture panel that enriches 186 known retinal disease genes, including 55 known RP genes, was developed. Targeted next-generation sequencing was performed for a cohort of 82 unrelated RP cases from Northern Ireland, including 46 simplex cases and 36 familial cases. Disease-causing mutations were identified in 49 probands, including 28 simplex cases and 21 familial cases, achieving a solving rate of 60 %. In total, 65 pathogenic mutations were found, and 29 of these were novel. Interestingly, the molecular information of 12 probands was neither consistent with their initial inheritance pattern nor clinical diagnosis. Further clinical reassessment resulted in a refinement of the clinical diagnosis in 11 patients. This is the first study to apply next-generation sequencing-based, comprehensive molecular diagnoses to a large number of RP probands from Northern Ireland. Our study shows that molecular information can aid clinical diagnosis, potentially changing treatment options, current family counseling and management.

Within-host evolution of Burkholderia pseudomallei over a twelve-year chronic carriage infection

UNLABELLED: Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR, an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host.

IMPORTANCE: Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.

Next-generation sequencing: a change of paradigm in molecular diagnostic validation

Next-generation sequencing (NGS) is beginning to show its full potential for diagnostic and therapeutic applications. In particular, it is enunciating its capacity to contribute to a molecular taxonomy of cancer, to be used as a standard approach for diagnostic mutation detection, and to open new treatment options that are not exclusively organ-specific. If this is the case, how much validation is necessary and what should be the validation strategy, when bringing NGS into the diagnostic/clinical practice? This validation strategy should address key issues such as: what is the overall extent of the validation? Should essential indicators of test performance such as sensitivity of specificity be calculated for every target or sample type? Should bioinformatic interpretation approaches be validated with the same rigour? What is a competitive clinical turnaround time for a NGS-based test, and when does it become a cost-effective testing proposition? While we address these and other related topics in this commentary, we also suggest that a single set of international guidelines for the validation and use of NGS technology in routine diagnostics may allow us all to make a much more effective use of resources.

Perception and Automatic Recognition of Laughter from Whole-Body Motion: Continuous and Categorical Perspectives

Despite its importance in social interactions, laughter remains little studied in affective computing. Intelligent virtual agents are often blind to users’ laughter and unable to produce convincing laughter themselves. Respiratory, auditory, and facial laughter signals have been investigated but laughter-related body movements have received less attention. The aim of this study is threefold. First, to probe human laughter perception by analyzing patterns of categorisations of natural laughter animated on a minimal avatar. Results reveal that a low dimensional space can describe perception of laughter “types”. Second, to investigate observers’ perception of laughter (hilarious, social, awkward, fake, and non-laughter) based on animated avatars generated from natural and acted motion-capture data. Significant differences in torso and limb movements are found between animations perceived as laughter and those perceived as non-laughter. Hilarious laughter also differs from social laughter. Different body movement features were indicative of laughter in sitting and standing avatar postures. Third, to investigate automatic recognition of laughter to the same level of certainty as observers’ perceptions. Results show recognition rates of the Random Forest model approach human rating levels. Classification comparisons and feature importance analyses indicate an improvement in recognition of social laughter when localized features and nonlinear models are used.

Characterisation of a novel panel of polymorphic microsatellite loci for the liver fluke, Fasciola hepatica, using a next generation sequencing approach

The liver fluke, Fasciola hepatica is an economically important pathogen of sheep and cattle and has been described by the WHO as a re-emerging zoonosis. Control is heavily reliant on the use of drugs, particularly triclabendazole and as a result resistance has now emerged. The population structure of F. hepatica is not well known, yet it can impact on host-parasite interactions and parasite control with drugs, particularly regarding the spread of triclabendazole resistance. We have identified 2448 potential microsatellites from 83Mb of F. hepatica genome sequence using msatfinder. Thirty-five loci were developed and optimised for microsatellite PCR, resulting in a panel of 15 polymorphic loci, with a range of three to 15 alleles. This panel was validated on genomic DNA from 46 adult F. hepatica; 38 liver flukes sourced from a Northwest abattoir, UK and 8 liver flukes from an established isolate (Shrewsbury; Ridgeway Research). Evidence for null alleles was found at four loci (Fh_1, Fh_8, Fh_13 and Fh_14), which showed markedly higher levels of homozygosity than the remaining 11 loci. Of the 38 liver flukes isolated from cattle livers (n=10) at the abattoir, 37 genotypes were identified. Using a multiplex approach all 15 loci could be amplified from several life cycle stages that typically yield low amounts of DNA, including metacercariae, the infective life cycle stage present on pasture, highlighting the utility of this multiplex microsatellite panel. This study reports the largest panel of microsatellite markers available to date for population studies of F. hepatica and the first multiplex panel of microsatellite markers that can be used for several life cycle stages.

The whole song is greater than the sum of its parts: Local and structural features in music listening

Four experiments reported here demonstrate the importance of structural as well as local features in listening to contemporary popular music. Experiment 1 established that listeners without formal musical training regard as salient the formal structure that links individual sections of songs. When asked to listen to and assemble the individual sections of unfamiliar contemporary songs to form new compositions, participants positioned the sections in ways consistent with the true structure of the music. In Experiment 2, participants were provided with only the song lyrics with which to arrange the individual sections of contemporary songs. It was found that in addition to musical features
studied in Experiment 1, lyrical content of contemporary music also acts as a strong cue to a song’s formal structure. Experiments 3 and 4 revealed that listeners’ enjoyment of music is influenced both by structural features and local features of music, which were carried by the individual song sections.
The influence of structural features on music listening was most apparent over repeated hearings. In Experiment 4, listeners’ liking for contemporary music followed an inverted U-shape trend with repeated exposure, in which liking for music took a downward turn after just four repeated hearings. In contrast, liking for restructured music increased with repeated hearings and almost eliminated an initial negative effect of restructuring by the sixth hearing. In sum, our findings demonstrate that structural features as well as local features of contemporary music are salient and important to
listeners.

How should teaching on whole person medicine, including spiritual issues, be delivered in the undergraduate medical curriculum in the United Kingdom?

Background

Although the General Medical Council recommends that United Kingdom medical students are taught ‘whole person medicine’, spiritual care is variably recognised within the curriculum. Data on teaching delivery and attainment of learning outcomes is lacking. This study ascertained views of Faculty and students about spiritual care and how to teach and assess competence in delivering such care.

A questionnaire comprising 28 questions exploring attitudes to whole person medicine, spirituality and illness, and training of healthcare staff in providing spiritual care was designed using a five-point Likert scale. Free text comments were studied by thematic analysis. The questionnaire was distributed to 1300 students and 106 Faculty at Queen’s University Belfast Medical School.

351 responses (54 staff, 287 students; 25 %) were obtained. >90 % agreed that whole person medicine included physical, psychological and social components; 60 % supported inclusion of a spiritual component within the definition. Most supported availability of spiritual interventions for patients, including access to chaplains (71 %), counsellors (62 %), or members of the patient’s faith community (59 %). 90 % felt that personal faith/spirituality was important to some patients and 60 % agreed that this influenced health. However 80 % felt that doctors should never/rarely share their own spiritual beliefs with patients and 67 % felt they should only do so when specifically invited. Most supported including training on provision of spiritual care within the curriculum; 40-50 % felt this should be optional and 40 % mandatory. Small group teaching was the favoured delivery method. 64 % felt that teaching should not be assessed, but among assessment methods, reflective portfolios were most favoured (30 %). Students tended to hold more polarised viewpoints but generally were more favourably disposed towards spiritual care than Faculty. Respecting patients’ values and beliefs and the need for guidance in provision of spiritual care were identified in the free-text comments.

Students and Faculty generally recognise a spiritual dimension to health and support provision of spiritual care to appropriate patients. There is lack of consensus whether this should be delivered by doctors or left to others. Spiritual issues impacting patient management should be included in the curriculum; agreement is lacking about how to deliver and assess.