Unique Capabilities of Genome Sequencing for Rare Disease Diagnosis.

Background: Causal variants underlying rare disorders may remain elusive even after expansive gene panels or exome sequencing (ES). Clinicians and researchers may then turn to genome sequencing (GS), though the added value of this technique and its optimal use remain poorly defined. We therefore investigated the advantages of GS within a phenotypically diverse cohort. Methods: GS was performed for 744 individuals with rare disease who were genetically undiagnosed. Analysis included review of single nucleotide, indel, structural, and mitochondrial variants. Results: We successfully solved 218/744 (29.3%) cases using GS, with most solves involving established disease genes (157/218, 72.0%). Of all solved cases, 148 (67.9%) had previously had non-diagnostic ES. We systematically evaluated the 218 causal variants for features requiring GS to identify and 61/218 (28.0%) met these criteria, representing 8.2% of the entire cohort. These included small structural variants (13), copy neutral inversions and complex rearrangements (8), tandem repeat expansions (6), deep intronic variants (15), and coding variants that may be more easily found using GS related to uniformity of coverage (19). Conclusion: We describe the diagnostic yield of GS in a large and diverse cohort, illustrating several types of pathogenic variation eluding ES or other techniques. Our results reveal a higher diagnostic yield of GS, supporting the utility of a genome-first approach, with consideration of GS as a secondary or tertiary test when higher-resolution structural variant analysis is needed or there is a strong clinical suspicion for a condition and prior targeted genetic testing has been negative.

Addressing complex pharmacy consultations: methods used to develop a person-centred intervention to highlight alcohol within pharmacist reviews of medications.

BACKGROUND: Alcohol is challenging to discuss, and patients may be reluctant to disclose drinking partly because of concern about being judged. This report presents an overview of the development of a medications review intervention co-produced with the pharmacy profession and with patients, which breaks new ground by seeking to give appropriate attention to alcohol within these consultations. METHODS: This intervention was developed in a series of stages and refined through conceptual discussion, literature review, observational and interview studies, and consultations with advisory groups. In this study we reflect on this process, paying particular attention to the methods used, where lessons may inform innovations in other complex clinical consultations. RESULTS: Early work with patients and pharmacists infused the entire process with a heightened sense of the complexity of consultations in everyday practice, prompting careful deliberation on the implications for intervention development. This required the research team to be highly responsive to both co-production inputs and data gathered in formally conducted studies, and to be committed to working through the implications for intervention design. The intervention thus evolved significantly over time, with the greatest transformations resulting from patient and pharmacist co-design workshops in the second stage of the process, where pharmacists elaborated on the nature of the need for training in particular. The original research plans provided a helpful structure, and unanticipated issues for investigation emerged throughout the process. This underscored the need to engage dynamically with changing contexts and contents and to avoid rigid adherence to any early prescribed plan. CONCLUSIONS: Alcohol interventions are complex and require careful developmental research. This can be a messy enterprise, which can nonetheless shed new insights into the challenges involved in optimising interventions, and how to meet them, if embraced with an attitude of openness to learning. We found that exposing our own research plans to scrutiny resulted in changes to the intervention design that gained the confidence of different stakeholders. Our understanding of the methods used, and their consequences, may be bounded by the person-centred nature of this particular intervention.

A pilot cluster randomised trial of the medicines and alcohol consultation (MAC): an intervention to discuss alcohol use in community pharmacy medicine review services.

BACKGROUND: Alcohol interventions are important to the developing public health role of community pharmacies. The Medicines and Alcohol Consultation (MAC) is a new intervention, co-produced with community pharmacists (CPs) and patients, which involves a CP practice development programme designed to integrate discussion of alcohol within existing NHS medicine review services. We conducted a pilot trial of the MAC and its delivery to investigate all study procedures to inform progression to a definitive trial. METHODS: This cluster pilot RCT was conducted in 10 community pharmacies in Yorkshire, UK, with a CP from each who regularly conducted Medicine Use Review (MUR) and New Medicine Service (NMS) consultations. Randomisation was conducted using a secure remote randomisation service. Intervention CPs (n = 5) were trained to deliver the MAC in MUR/NMS consultations. Control CPs (n = 5) provided these services as usual. Consecutive MUR/NMS patients were asked by CPs to participate, screened for eligibility (consumption of alcohol at least twice per week), and baseline data collected for those eligible. A two-month follow-up telephone interview was conducted. Blinding of CPs was not possible, but patients were blinded to the alcohol focus of the trial. Primary outcomes were total weekly UK units (8 g of ethanol per unit) of alcohol consumption in the week prior to follow-up, and confidence in medications management. Trial procedures were assessed by recruitment, attrition, and follow-up rates. RESULTS: 260 patients were approached by CPs to take part in the trial, 68% (n = 178) were assessed for eligibility and 30% (n = 54) of these patients were eligible. Almost all eligible patients (n = 51; 94%) consented to participate, of whom 92% (n = 47) were followed-up at 2 months; alcohol consumption was lower in the intervention arm and confidence in medication management reduced slightly for both groups. Exploration of recall issues at follow-up showed a high level of agreement between a two-item quantity/frequency measure and 7-day guided recall of alcohol consumption. CONCLUSIONS: The pilot trial demonstrates the feasibility of implementing the MAC in community pharmacy and trial recruitment and data collection procedures. However, decommissioning of MURs means that it is not possible to conduct a definitive trial of the intervention in this service. TRIAL REGISTRATION: ISRCTN57447996.

Costello syndrome: Clinical phenotype, genotype, and management guidelines.

Costello syndrome (CS) is a RASopathy caused by activating germline mutations in HRAS. Due to ubiquitous HRAS gene expression, CS affects multiple organ systems and individuals are predisposed to cancer. Individuals with CS may have distinctive craniofacial features, cardiac anomalies, growth and developmental delays, as well as dermatological, orthopedic, ocular, and neurological issues; however, considerable overlap with other RASopathies exists. Medical evaluation requires an understanding of the multifaceted phenotype. Subspecialists may have limited experience in caring for these individuals because of the rarity of CS. Furthermore, the phenotypic presentation may vary with the underlying genotype. These guidelines were developed by an interdisciplinary team of experts in order to encourage timely health care practices and provide medical management guidelines for the primary and specialty care provider, as well as for the families and affected individuals across their lifespan. These guidelines are based on expert opinion and do not represent evidence-based guidelines due to the lack of data for this rare condition.

Polymicrogyria in association with hypoglycemia points to mutation in the mTOR pathway.

We report a 16-month-old male with congenital megalencephaly, polymicrogyria and persistent hypoglycemia caused by a mosaic PIK3CA pathogenic variant. Hypoinsulinaemic, hypoketotic hypoglycaemia is a rare complication of pathogenic variants in the PI3K-AKT-mTOR pathway genes including AKT2, AKT3, CCND2, PIK3R2 and PIK3CA, and has been identified in a PIK3CA mutant mouse model. Our case highlights the importance of considering PI3K-AKT-mTOR pathway variants as a cause for megalencephaly and cortical malformation when the phenotype includes hypoglycaemia. Recognizing the association of hypoglycemia with PI3K-AKT-mTOR pathway variants can provide a clue to the genetic basis of the cortical malformation. Patients with megalencephaly and a cortical malformation may be considered at risk of hypoglycaemia and monitored accordingly, at least until a PI3K-AKT-mTOR pathway variant has been excluded.

Program Evaluation of the Inner South Community Health Oral Health Program for Priority Populations.

BACKGROUND: This study evaluated an Oral Health Program for Priority Populations (OHPPP) in the Inner South of Melbourne, Victoria. METHODS: Social Ecological Theory and the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework informed the study. It involved administering satisfaction questionnaires to 29 clients and conducting eight in-depth interviews and four focus group discussions with either clients, health care workers or partner agencies. RESULTS: Most (92%) clients surveyed felt more positive about their health as a result of the OHPPP. Three themes emerged from the qualitative analyses: 1) good oral health is central to improving general health; 2) the OHPPP is valuable; and 3) there are difficulties in implementing the OHPPP. CONCLUSIONS: Clients and service providers view the OHPPP as accessible and rewarding. Our findings point to the need for policies that recognise the greater treatment needs of disadvantaged populations and that streamline the provision of their dental care.

Mandibulofacial Dysostosis with Microcephaly: Mutation and Database Update.

Mandibulofacial dysostosis with microcephaly (MFDM) is a multiple malformation syndrome comprising microcephaly, craniofacial anomalies, hearing loss, dysmorphic features, and, in some cases, esophageal atresia. Haploinsufficiency of a spliceosomal GTPase, U5-116 kDa/EFTUD2, is responsible. Here, we review the molecular basis of MFDM in the 69 individuals described to date, and report mutations in 38 new individuals, bringing the total number of reported individuals to 107 individuals from 94 kindreds. Pathogenic EFTUD2 variants comprise 76 distinct mutations and seven microdeletions. Among point mutations, missense substitutions are infrequent (14 out of 76; 18%) relative to stop-gain (29 out of 76; 38%), and splicing (33 out of 76; 43%) mutations. Where known, mutation origin was de novo in 48 out of 64 individuals (75%), dominantly inherited in 12 out of 64 (19%), and due to proven germline mosaicism in four out of 64 (6%). Highly penetrant clinical features include, microcephaly, first and second arch craniofacial malformations, and hearing loss; esophageal atresia is present in an estimated ∼27%. Microcephaly is virtually universal in childhood, with some adults exhibiting late "catch-up" growth and normocephaly at maturity. Occasionally reported anomalies, include vestibular and ossicular malformations, reduced mouth opening, atrophy of cerebral white matter, structural brain malformations, and epibulbar dermoid. All reported EFTUD2 mutations can be found in the EFTUD2 mutation database (http://databases.lovd.nl/shared/genes/EFTUD2).

Approaches to study in undergraduate nursing students in regional Victoria, Australia.

In developmental research to devise a strategy to identify students who may benefit from assistance with learning habits, approaches to study were explored in undergraduate nursing students (n=122) enrolled in a compulsory first-year course in physiology at a regional Australian university. The course constituted 30 credits (25%) of their first year of study. Using the Approaches and Study Skills Inventory (ASSIST), students were identified as adopting a deep (n = 38, 31%), strategic (n = 30, 25%), or a surface (n = 54, 44%) approach to study. Internal consistency (Cronbach's alpha [α]) for deep, strategic, and surface was 0.85, 0.87, and 0.76, respectively. Subsequently, a cluster analysis was done to identify two groupings: a "surface" group (n = 53) and a "deep/strategic" group (n = 69). The surface group scored lower in deep (33.28 ± 6.42) and strategic (39.36 ± 6.79) approaches and higher in the surface (46.96 ± 9.57) approach. Conversely, the deep/strategic group scored 46.10 ± 6.81, 57.17 ± 7.81, and 41.87 ± 6.47 in deep, strategic, and surface styles, respectively. This application of the ASSIST questionnaire and cluster analysis thus differentiated students adopting a surface approach to study. This strategy may enable educators to target resources, for example additional tutorial opportunities, peer-assisted study support, and tutor-led seminar sessions aimed at encouraging students to adopt a less superficial approach to study.

Making sense of complex electronic records: socio-technical design in social care.

Dealing with complex electronic documentation is an integral part of much contemporary professional work. In this paper, we address the design of electronic records for social care professionals in the UK. Recent reforms in UK child welfare have followed a top-down, managerial approach emphasizing conformance to standard processes. The vicissitudes of a major national IT project, the Integrated Children's System, show the limitations of this approach, in particular the detrimental effect it has had on professional autonomy. Following in the foot-steps of Ken Eason, we argue that socio-technical design, by focussing on innovative applications of technology to support users (rather than the interests of the bureaucracy) offers a more promising alternative. A user-centred design exercise is presented to illustrate this approach in action. A novel interface was developed for handling the heterogeneous bundle of documents which make up the social care record, helping social workers make better sense of case-files. The prototype draws on the metaphor of the dining-room table as a way of overcoming the limitations of the computer display. We conclude that socio-technical thinking engenders a shift in mind-set, opening up a radically different design space compared to current design orthodoxy.

Beyond bureaucracy: emerging trends in social care informatics.

Existing information technology systems in much of UK social care have been designed to serve the interests of the bureaucracy rather than supporting professional practice or improving services to the public. The ill-starred Integrated Children's System in statutory children's services is typical. The Integrated Children's System is a system for form-filling, micro-managing professional practice through an enforced regime of standard processes and time scales. In this article, we argue against this dominant design. We provide several examples where technology has enabled alternative modes of support for professional work, based on socio-technical principles. One such system is Patchwork, which describes itself as a 'Facebook for Social Work'; its aim is to support multi-professional teams working with vulnerable families.

Effects of MHRA drug safety advice on time trends in prescribing volume and indices of clinical toxicity for quinine.

AIMS: To ascertain the effects of the Medicines and Healthcare products Regulatory Agency's (MHRA) safety update in June 2010 on the volume of prescribing of quinine and on indices of quinine toxicity. METHODS: We analysed quarterly primary care total and quinine prescribing data for England and quinine prescribing volume for individual Primary Care Trusts in the North East of England from 2007/8 to 2011/12 obtained from the ePACT.net database. We also analysed quinine toxicity enquiries to the National Poisons Information Service (NPIS) via Toxbase(®) and by telephone between 2004/5 and 2011/12. Joinpoint regression and Pearson's correlation tests were used to ascertain changes in trends in prescribing and indices of toxicity and associations between prescribing and indices of toxicity, respectively. RESULTS: Total prescribing continued to increase, but annual growth in quinine prescribing in England declined from 6.0 to -0.6% following the MHRA update [difference -0.04 (95% confidence interval -0.07 to -0.01) quinine prescriptions per 100 patients per quarter, P = 0.0111]. Much larger reductions were observed in Primary Care Trusts that introduced comprehensive prescribing reviews. The previously increasing trend in Toxbase(®) quinine searches was reversed [difference -19.76 (95% confidence interval -39.28 to -9.20) user sessions per quarter, P = 0.0575]. Telephone enquiries to NPIS for quinine have declined, with stabilization of the proportion of moderate to severe cases of quinine poisoning since the update. CONCLUSIONS: The MHRA advice was followed by limited reductions in the growth in quinine prescribing and in indicators of quinine overdose and toxicity. Quinine prescribing, however, remains common, and further efforts are needed to reduce availability and use.

Electronic referrals: what matters to the users.

BACKGROUND: Between September 2010 and May 2011 we evaluated three implementations of electronic referral (eReferral) systems at Hutt Valley, Northland and Canterbury District Health Boards in New Zealand. METHODS: Qualitative and quantitative data were gathered through project documentation, database records and stakeholder interviews. This paper reports on the user perspectives based on interviews with 78 clinical, management and operational stakeholders in the three regions. Themes that emerge across the regions are compared and synthesised. Interviews focused on pre-planned domains including quality of referral, ease of use and patient safety, but agendas were adapted progressively to elaborate and triangulate on themes emerging from earlier interviews and to clarify indications from analysis of database records. RESULTS AND DISCUSSION: The eReferral users, including general practitioners, specialists and administrative staff, report benefits in the areas of: (1) availability and transparency of referral-related data; (2) work transformation; (3) improved data quality and (4) the convenience of auto-population from the practice management system into the referral forms. eReferral provides enhanced visibility of referral data and status within the limits of the implementation (which only goes to the hospital door in some cases). Users in all projects indicated the desire to further exploit IT to enhance two-way communication between community and hospital. Reduced administrative handling is a clear work transformation benefit with mixed feedback regarding clinical workload impact. Innovations such as GP eReferral triaging teams illustrate the further potential for workflow transformation. Consistent structure in eReferrals, as well as simple legibility, enhances data quality. Efficiency and completeness is provided by auto-population of forms from system data, but opens issues around data accuracy. All three projects highlight the importance of user involvement in design, implementation and refinement. In keeping with this, Canterbury utilises a systematic pathway definition process that brings together GPs and specialist to debate and agree on the local management of a condition. User feedback exposes many opportunities for improving usability. STUDY LIMITATIONS: The findings are based on individual experiences accounted by participating stakeholders; the risk of bias is mitigated, however, by triangulation across three distinct implementations of eReferrals. Quantitative follow-up on key outstanding issues, notably impact of structured eReferral forms on GP time to write a referral, is warranted. CONCLUSION: Key eReferral users include clinicians on both ends of the referral process as well as the administrative staff. User experience in three eReferral projects has shown that they particularly appreciate improvement of referral visibility, as well as information quality; promising workflow transformations have been achieved in some places. Auto-population of forms leads to opportunities, and issues, that are prompting further attention to data quality. While the importance of user feedback should be obvious, it is not universal to seek it or to provide resources to effectively follow up with improvements driven by such feedback. To maximise benefits, innovative health IT projects must take an iterative approach guided by ongoing user experience.

Water quality targets and maintenance of valued landscape character - experience in the Axe catchment, UK.

The Water Framework Directive (WFD) (Directive 2000/60/EC) requires new ecological standards for rivers, lakes and coastal waters by 2015. In the United Kingdom the English Catchment Sensitive Farming Initiative has identified 40 catchments which are at risk of failing the European Commission WFD targets for good ecological status of water bodies because of a range of issues. The river Axe catchment situated in south-west England, with a mixture of diffuse and point sources of pollution, is one of these priority sites, as intensive dairy farming and cultivation of high risk crops (maize) cause problems with enhanced suspended sediment, nitrate and phosphorus levels in the river. Much of the Axe is under national and county landscape designations, making land use or management measures taken to achieve river status sensitive to these designations. For the purpose of this research the Soil and Water Assessment Tool (SWAT-2005) was used. The baseline scenario was based on field observation and interviews with the Environment Agency and farmers; it was run with and without point sources. Three different mitigation scenarios, designed to maintain the landscape of the catchment, were then tested. Field buffer strips (FBS), extensive land use management (EXT) and sheep land use management (SHP), were used to assess the effectiveness of the measures in reducing nutrient loads in the river Axe, UK. Management scenarios reduced the average annual loads at the main catchment outlet by 21.2% (FBS), 37.3% (EXT) and 45.0% (SHP), for total nitrogen and 47.7% (FBS), 60.6% (EXT) and 62.4% (SHP) for total phosphorus. The results of this study suggest that there may be a fundamental incompatibility between the delivery of WFD targets and the maintenance of viable agricultural systems necessary to maintain landscapes which are highly valued for their aesthetic, recreational and economic value.

Using artificial fluorescent particles as tracers of livestock wastes within an agricultural catchment.

Evidence for the movement of agricultural slurry and associated pollutants into surface waters is often anecdotal, particularly with relation to its 'particulate' components which receive less attention than 'bio-available' soluble phases. To assess the extent of movement of slurry particles artificial fluorescent particles were mixed with slurry and applied to a field sub-catchment within a headwater catchment. Particles were 2-60 µm in diameter and two different densities, 2.7 and 1.2 g cm(-3) representing 'inorganic' and 'organic' material. Water samples from the field and catchment outlet were collected during two storm events following slurry application and analysed for particle and suspended sediment concentrations (SSC). SSC from the field and catchment outlet always formed clockwise hysteresis loops indicating sediment exhaustion and particles of the two densities were always found to be positively correlated. Particles from the field formed clockwise hysteresis loops during the first discharge event after slurry application, but anti-clockwise hysteresis loops during the second monitored event which indicated a depletion of readily mobilisable particles. Particles from the catchment outlet always formed anticlockwise hysteresis loops. Particle size became finer spatially, between field and catchment outlet, and temporally, between successive storm events. The results indicate that slurry particles may be readily transported within catchments but that different areas may contribute to pollutant loads long after the main peak in SSC has passed. The density of the particles did not appear to have any effect on particle transport however the size of the particles may play a more important role in the 2-60 µm range.

The hydrological response of heavy clay grassland soils to rainfall in south-west England using delta2H.

Stable isotopes of water have been previously used in catchment studies to separate rain-event water from pre-event groundwater. However, there are a lack of studies at the smaller scale looking at the separation of event water from pre-event water. This is particularly relevant for heavy clay soil systems through which the movement of water is uncertain but is thought to be rainwater-dominated. The data presented here were collected at a rural site in the south-west of England. The historic rainfall at the site was isotopically varied but similar to the global meteoric water line, with annual weighted means of -37 per thousand for delta(2)H and -5.7 per thousand for delta(18)O and with no seasonal variation. Drainage was sampled from the inter-flow (surface runoff + lateral through-flow) and drain-flow (55 cm deep mole drains) pathways of two 1 ha lysimeters during two rainfall events, which had delta(2)H values of -68 per thousand and -92 per thousand, respectively. The delta(2)H values of the lysimeter drainage water suggest that there was no contribution of event water during the first, small discharge (Q) event; however, the second larger event did show isotopic variation in delta(2)H values negatively related to Q indicating that rainwater was contributing to Q. A hydrograph separation indicated that only 49-58% of the inter-flow and 18-25% of the drain-flow consisted of event water. This was surprising given that these soil types are considered retentive of soil water. More work is needed on heavy clay soils to understand better the nature of water movement from these systems.

Assessing multiple novel tracers to improve the understanding of the contribution of agricultural farm waste to diffuse water pollution.

A study was undertaken on drained and undrained 1 ha grassland lysimeters to assess the effectiveness of multiple novel tracing techniques in understanding how agricultural slurry waste moves from land to water. Artificial fluorescent particles designed to mimic the size and density of organic slurry particles were found to move off the grassland via inter-flow (surface + lateral through-flow) and drain-flow. Where both pathways were present the drains carried the greater number of particles. The results of the natural fluorescence and δ13C of water samples were inconclusive. Natural fluorescence was higher from slurry-amended lysimeters than from zero-slurry lysimeters, however, a fluorescence decay experiment suggested that no slurry signal should be present given the time between slurry application and the onset of drainage. The δ13C values of >0.7 microm and <0.7 microm material in drainage were varied and unrelated to discharge. The mean value of >0.7 microm δ13C in water from the drain-flow pathways was higher from the lysimeter which had received naturally enriched maize slurry compared to the lysimeter which received grass slurry indicating a contribution of slurry-derived material. Values of <0.7 microm δ13C from the same pathway, however, produced counter intuitive trends and may indicate that different fractions of the slurry have different δ13C values.

Outbreaks of Salmonella Typhimurium phage type 197 of multiple genotypes linked to an egg producer.

This paper describes outbreaks of Salmonella Typhimurium phage type 197 (STm197) linked to eggs from the farm of a single egg producer. Epidemiological and microbiological investigations (genotyping by multiple locus variable number tandem repeats analysis [MLVA]) identified outbreaks of STm197 with the same or closely-related MLVA profiles in a series of restaurants across Brisbane over 2 months. Environmental health investigations revealed that these restaurants were supplied with eggs from the same egg producer and that cross-contamination may have contributed to the outbreak. Environmental swabs taken from restaurant kitchens and the farm of the egg producer identified a number of salmonellas including STm197, many with MLVA profiles matching or closely related to the human strains from outbreak cases. A case-to-case comparison study showed a significant association between illness with 1 MLVA type and attending a restaurant during the 5 days before onset of illness (odds ratio [OR] 8.1, 95% confidence interval [CI] 1.8, 35.4). MLVA has become a valuable tool for S. Typhimurium surveillance and outbreak investigation. This outbreak further justifies the Commonwealth Government's decision to develop a draft national primary production and processing standard for eggs and egg products to address food safety risks posed by cracked and dirty eggs.