Addressing ethical and laboratory challenges for initiation of a rapid whole genome sequencing program.

Rapid whole genome sequencing (rapid WGS) is a powerful diagnostic tool that is becoming increasingly practical for widespread clinical use. However, protocols for its use are challenging to implement. A significant obstacle to clinical adoption is that laboratory certification requires an initial research development phase, which is constrained by regulations from returning results. Regulations preventing return of results have ethical implications in cases which might impact patient outcomes. Here, we describe our experience with the development of a rapid WGS research protocol, that balanced the requirements for laboratory-validated test development with the ethical needs of clinically relevant return of results.

Effective variant filtering and expected candidate variant yield in studies of rare human disease.

In studies of families with rare disease, it is common to screen for de novo mutations, as well as recessive or dominant variants that explain the phenotype. However, the filtering strategies and software used to prioritize high-confidence variants vary from study to study. In an effort to establish recommendations for rare disease research, we explore effective guidelines for variant (SNP and INDEL) filtering and report the expected number of candidates for de novo dominant, recessive, and autosomal dominant modes of inheritance. We derived these guidelines using two large family-based cohorts that underwent whole-genome sequencing, as well as two family cohorts with whole-exome sequencing. The filters are applied to common attributes, including genotype-quality, sequencing depth, allele balance, and population allele frequency. The resulting guidelines yield ~10 candidate SNP and INDEL variants per exome, and 18 per genome for recessive and de novo dominant modes of inheritance, with substantially more candidates for autosomal dominant inheritance. For family-based, whole-genome sequencing studies, this number includes an average of three de novo, ten compound heterozygous, one autosomal recessive, four X-linked variants, and roughly 100 candidate variants following autosomal dominant inheritance. The slivar software we developed to establish and rapidly apply these filters to VCF files is available at https://github.com/brentp/slivar under an MIT license, and includes documentation and recommendations for best practices for rare disease analysis.

The impact of hydraulic retention time on the performance of two configurations of anaerobic pond for municipal sewage treatment.

Anaerobic ponds have the potential to contribute to low carbon wastewater treatment, however are currently restricted by long hydraulic residence time (HRT) which leads to large land requirements. A two-stage anaerobic pond (SAP) design was trialled against a single-stage control (CAP) over four HRTs down to 0.5 days, to determine the lowest HRT at which the ponds could operate effectively. No statistical differences were observed in particulate removal between the ponds over all four HRTs, suggesting solids loading is not a critical factor in AP design. Significantly higher biogas production rates were observed in the SAP than the CAP at 1.5 d and 1.0 d HRT, and microbial community profiling suggests the two-stage design may be facilitating spatial separation of the anaerobic digestion process along reactor length. Hydrogenotrophic methanogensis dominated over aceticlastic, with acetate oxidisation a likely degradation pathway. Experimental tracer studies were compared to CFD simulations, with the SAP showing greater hydraulic efficiency, and differences more pronounced at shorter HRTs. Greater flow recirculation between baffles was observed in CFD velocity profiles, demonstrating baffles can dissipate preferential flow patterns and increase effective pond volume, especially at high flow rates. The study demonstrates the potential of APs to be operated at shorter HRTs in psychrophilic conditions, presenting an opportunity for use as pre-treatments (in place of septic tanks) and primary treatment for full wastewater flows. Two-stage designs should be investigated to separate the stages of the anaerobic digestion process by creating preferential conditions along the pond length.

No observed reduction of non-attendance rate in telehealth models of care.

Objective This study investigated the provision of public specialist out-patient services in Queensland delivered in traditional hospital settings (in person) or through a two-way synchronous videoconferencing session (telehealth). Rates of attendance between these delivery methods were compared to detect any difference in rates of non-attendance among patients. Methods An extract of all specialist out-patient appointments reported in Queensland Health's corporate patient administration systems between 1 July 2017 and 30 June 2018 was obtained (n = 2921702). Variables including how the service was delivered and whether the patient attended were captured for each event. Results No reduction in non-attendance was observed in the telehealth patient group (9.1%) compared with in-person service delivery (9.1% vs 7.9% respectively; = 113.56, P < 0.001, relative risk = 1.15). Discussion The study found no evidence that telehealth is effective at reducing rates of non-attendance in a specialist out-patient setting. This supports existing findings that most non-attendance is the result of forgetfulness or confusion with appointment details, to which telehealth appointments are also vulnerable. What is known about the topic? Non-attendance of out-patient appointments remains a persistent and costly problem for public and private providers of health services. Forgetting or being confused about appointment details are the most commonly reported reasons for patient non-attendance. What does this paper add? Telehealth models of care are increasingly being offered by health service providers, reducing travel requirements to all patients, particularly those in regional and remote settings. However, telehealth models of care do not address the most common reasons for patient non-attendance and telehealth patients are not less likely to miss their appointments. What are the implications for practitioners? Suggestions that telehealth models of care can reduce rates of non-attendance should be treated with caution by health service administrators and clinicians. More timely appointment reminders and easier processes to cancel or reschedule appointments remain the most effective techniques for reducing non-attendance.

Does the effectiveness of mutual aid depend on compatibility with treatment philosophies offered at residential rehabilitation services?

BACKGROUND: Residential rehabilitation treatment (including both Therapeutic Communities (TC) and non-TC rehabs) is a key component of service delivery for people seeking treatment for substance use disorders in Australia and globally. While mutual aid is often associated with better long-term outcomes, there is little evidence about whether inconsistencies between residential rehabilitation philosophies and particular types of mutual aid influence subsequent engagement and treatment outcomes. OBJECTIVE: To assess the uptake of mutual aid groups (12-step and other) on individuals leaving TC (n = 58) or non-TC (n = 78) residential treatment, and measure its impact on substance use outcomes. METHODS: Using secondary analysis of existing data, the current paper reports on 12-month outcomes from a prospective cohort study of 230 individuals entering specialist alcohol and other drug residential rehabilitation treatment in two Australian states. RESULTS: Participants who attended TC settings were more likely to attend non-spiritual mutual aid groups (i.e., SMART Recovery) than non-TC residents. Engaging in mutual aid groups was associated with significantly improved outcomes for the non-TC residents only, where it significantly predicted abstinence (OR = 5.8, CI = 1.5-18.46) and reduced frequency of use of participants' primary drug of concern (OR = 8.6, CI = 2.6-28.6). CONCLUSIONS/IMPORTANCE: Although 12-step is the most readily available and accessible form of mutual aid in Australia and benefited those attending non-TC residential rehabilitation, individuals exiting a TC program (whether they have completed treatment or not) may benefit from other forms of post-treatment recovery support, including alternative forms of peer-based support. The findings suggest treatment outcomes may be enhanced when the philosophies of residential treatment and post-discharge mutual aid are more compatible.

Orofacial cleft management by short-term surgical missions in South America: literature review.

There is a growing demand for surgical care in South America, particularly for patients with congenital orofacial clefts (OFCs). Short-term surgical missions (STSMs) have emerged as a means to deliver surgical expertise and alleviate this demand. The aim of this study was to review the quantity and quality of peer-reviewed reports on OFC repairs performed by STSMs in South America. A literature search was conducted using the PubMed, Embase, Web of Science, and SciELO databases. The search was limited to articles published in English and Spanish. Descriptive statistics were used for the data analysis. The search yielded 65 studies related to OFCs. Eight (12.3%) were selected for full-text review. Only five (7.7%) articles met the inclusion criteria. The diverse study designs and heterogeneous types of data assessment among the selected studies hindered a comparison between them. This review found a sparse number of publications pertaining to OFC missions to South America. The articles that were included demonstrated inconsistencies in reporting patient care data. There is a need for a more efficient, streamlined method of reporting humanitarian missions for OFC repairs in order for healthcare professionals to fulfill research and ethical obligations and offer the best practices in patient-centered care.

Genotype-Phenotype Correlations of Glucose-6-Phosphate-Deficient Variants Throughout an Activity Distribution.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked disorder that may manifest as neonatal jaundice or acute hemolytic anemia. Quantitative assessment of G6PD activity in erythrocytes is required to definitively diagnose a deficiency. Most males and homozygous females have low enzyme activities, whereas heterozygous females may have a range of activities. We sought to examine G6PD genotype-phenotype associations to identify an activity cutoff above which G6PD deficiency is unlikely. METHODS: Ninety-five residual samples were randomly selected to represent the various regions of a G6PD activity distribution. DNA was isolated from the leukocyte fraction and sequenced using the Sanger method. ROC curves were used to establish cutoffs. RESULTS: Thirteen variant alleles were identified, including 1 not previously reported. In the very deficient activity range, we found males and homozygous females of both class II and III variants. In the deficient category, we found predominantly class III males and heterozygous females. The presumed deficient category contained class III and IV variants and nonvariants. An activity cutoff of <7.85 U/g hemoglobin (Hb) was 100% sensitive and 94% specific for identifying a G6PD-deficient male, and a cutoff of <8.95 U/g Hb was 90% sensitive and 82% specific for a deficient female. CONCLUSIONS: The observed activity groupings were not because of a particular variant class. Cutoffs to identify the presence of a deficiency variant for males and females may be useful when trying to decide whether to recommend genetic analysis.

Pulmonary veno-occlusive disease: Two children with gradual disease progression.

Pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis are rare forms of pulmonary vascular disease. We report two cases of affected children who had evidence of pulmonary hypertension 3-5 years before developing radiographic findings of pulmonary veno-occlusive disease or pulmonary capillary hemangiomatosis. Both patients experienced a moderate decrease in pulmonary arterial pressure during acute vasodilator testing. Both patients experienced an improvement in six-minute walk performance without an increase in pulmonary edema when treated with targeted therapy for pulmonary hypertension. In some patients, pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis may progress slowly over a period of months to years. A favorable acute vasodilator response may identify patients who will tolerate, and demonstrate transient clinical improvement with, medical therapy.

EIF2AK4 Mutations in Patients Diagnosed With Pulmonary Arterial Hypertension.

BACKGROUND: Differentiating pulmonary venoocclusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH) from idiopathic pulmonary arterial hypertension (IPAH) or heritable pulmonary arterial hypertension (HPAH) is important clinically. Mutations in eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) cause heritable PVOD and PCH, whereas mutations in other genes cause HPAH. The aim of this study was to describe the frequency of pathogenic EIF2AK4 mutations in patients diagnosed clinically with IPAH or HPAH. METHODS: Sanger sequencing and deletion/duplication analysis were performed to detect mutations in the bone morphogenetic protein receptor type II (BMPR2) gene in 81 patients diagnosed at 30 North American medical centers with IPAH (n = 72) or HPAH (n = 9). BMPR2 mutation-negative patients (n = 67) were sequenced for mutations in four other genes (ACVRL1, ENG, CAV1, and KCNK3) known to cause HPAH. Patients negative for mutations in all known PAH genes (n = 66) were then sequenced for mutations in EIF2AK4. We assessed the pathogenicity of EIF2AK4 mutations and reviewed clinical characteristics of patients with pathogenic EIF2AK4 mutations. RESULTS: Pathogenic BMPR2 mutations were identified in 8 of 72 (11.1%) patients with IPAH and 6 of 9 (66.7%) patients with HPAH. A novel homozygous EIF2AK4 mutation (c.257+4A>C) was identified in 1 of 9 (11.1%) patients diagnosed with HPAH. The novel EIF2AK4 mutation (c.257+4A>C) was homozygous in two sisters with severe pulmonary hypertension. None of the 72 patients with IPAH had biallelic EIF2AK4 mutations. CONCLUSIONS: Pathogenic biallelic EIF2AK4 mutations are rarely identified in patients diagnosed with HPAH. Identification of pathogenic biallelic EIF2AK4 mutations can aid clinicians in differentiating HPAH from heritable PVOD or PCH.

Infantile pulmonary capillary haemangiomatosis: a lethal form of pulmonary hypertension.

We describe the cases of two children who both presented in infancy with recurrent severe pulmonary hypertensive crises. Exhaustive clinical work-up failed to identify an underlying aetiology. The patients had no clinical response to steroids, immunoglobulins, or pulmonary vasodilators. Post-mortem examination revealed extensive invasive pulmonary capillary haemangiomatosis. There was no evidence of pulmonary venous occlusive disease. Given the lethal nature of this condition, early consideration of referral to a lung transplant centre should be considered in selected patients.

Associations between social identity diversity, compatibility, and recovery capital amongst young people in substance use treatment.

This study explored associations between group memberships and recovery capital amongst 20 young adults aged 18 to 21 years in residential alcohol and drug treatment. METHOD: Participants completed an interviewer administered research interview based on measures of recovery capital and a social networks assessment mapping group memberships, group substance use, and relationships between groups. RESULTS: Higher personal and social recovery capital was associated with lower diversity of group memberships, a higher number of positive links between groups, and greater compatibility of lower substance-using groups with other groups in the network. Higher compatibility of heavier-using groups was also associated with having a higher number of negative, antagonistic ties between groups. CONCLUSIONS: These findings indicate that it is higher compatibility of a lower substance-using social identity and lower-using group memberships that contributes to recovery capital. Further, positive ties between groups and lower diversity of group memberships appear to be key aspects in how multiple social identities that are held by young adults relate to personal and social recovery capital.

Social identity, social networks and recovery capital in emerging adulthood: A pilot study.

BACKGROUND: It has been argued that recovery from substance dependence relies on a change in identity, with past research focused on 'personal identity'. This study assessed support for a social identity model of recovery in emerging adults through examining associations between social identity, social networks, recovery capital, and quality of life. METHODS: Twenty participants aged 18-21 in residential treatment for substance misuse were recruited from four specialist youth drug treatment services - three detoxification facilities and one psychosocial rehabilitation facility in Victoria, Australia. Participants completed a detailed social network interview exploring the substance use of groups in their social networks and measures of quality of life, recovery capital, and social identity. RESULTS: Lower group substance use was associated with higher recovery capital, stronger identification with non-using groups, and greater importance of non-using groups in the social network. Additionally, greater identification with and importance of non-using groups were associated with better environmental quality of life, whereas greater importance conferred on using groups was associated with reduced environmental quality of life. CONCLUSIONS: Support was found for the role of social identity processes in reported recovery capital and quality of life. Future research in larger, longitudinal samples is required to improve understanding of social identity processes during treatment and early recovery and its relationship to recovery stability.

Pulmonary Arterial Hypertension: A Current Perspective on Established and Emerging Molecular Genetic Defects.

Pulmonary arterial hypertension (PAH) is an often fatal disorder resulting from several causes including heterogeneous genetic defects. While mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are the single most common causal factor for hereditary cases, pathogenic mutations have been observed in approximately 25% of idiopathic PAH patients without a prior family history of disease. Additional defects of the transforming growth factor beta pathway have been implicated in disease pathogenesis. Specifically, studies have confirmed activin A receptor type II-like 1 (ACVRL1), endoglin (ENG), and members of the SMAD family as contributing to PAH both with and without associated clinical phenotypes. Most recently, next-generation sequencing has identified novel, rare genetic variation implicated in the PAH disease spectrum. Of importance, several identified genetic factors converge on related pathways and provide significant insight into the development, maintenance, and pathogenetic transformation of the pulmonary vascular bed. Together, these analyses represent the largest comprehensive compilation of BMPR2 and associated genetic risk factors for PAH, comprising known and novel variation. Additionally, with the inclusion of an allelic series of locus-specific variation in BMPR2, these data provide a key resource in data interpretation and development of contemporary therapeutic and diagnostic tools.

When to offer genetic testing for pulmonary arterial hypertension.

Genetic testing is poised to play a greater role in the diagnosis and management of pulmonary arterial hypertension (PAH). Physicians who manage PAH should know the heritable PAH phenotypes, inheritance patterns, and responsible genes. They also should know indications, potential risks and benefits, and the issues surrounding genetic counselling and testing for patients with PAH.

Extracorporeal membrane modality conversions.

We report the case of a patient with cardiovascular and respiratory failure due to severe anaphylaxis requiring multiple extracorporeal membrane oxygenation (ECMO) cannulation strategies to provide adequate oxygen delivery and ventilatory support during a period of rapid physiological change. ECMO provides partial or complete support of oxygenation-ventilation and circulation. The choice of which ECMO modality to use is governed by anatomical (vessel size, cardiovascular anatomy and previous surgeries) and physiological (respiratory and/or cardiac failure) factors. The urgency with which ECMO needs to be implemented (emergency cardiopulmonary resuscitation (eCPR), urgent, elective) and the institutional experience will also influence the type of ECMO provided. Here we describe a 12-year-old schoolgirl who, having been resuscitated with peripheral veno-venous (VV) ECMO for severe hypoxemia due to status asthmaticus in the setting of acute anaphylaxis, required escalation to peripheral veno-arterial (VA) ECMO for precipitous cardiovascular deterioration. Insufficient oxygen delivery for adequate cellular metabolic function and possible cerebral hypoxia due to significant differential hypoxia necessitated ECMO modification. After six days of central (transthoracic) VA ECMO support and 21 days of intensive care unit (ICU) care, she made a complete recovery with no neurological sequelae. The use of ECMO support warrants careful consideration of the interplay of a patient's pathophysiology and extracorporeal circuit dynamics. Particular emphasis should be placed on the potential for mismatch between cardiovascular and respiratory support as well as the need to meet metabolic demands through adequate cerebral, coronary and systemic oxygenation. Cannulation strategies occasionally require alteration to meet and anticipate the patient's evolving needs.

Genetics of pulmonary hypertension.

PURPOSE OF REVIEW: The identification of the genetic basis for heritable predisposition to pulmonary arterial hypertension (PAH) has altered the clinical and research landscape for PAH patients and their care providers. This review aims to describe the genetic discoveries and their impact on clinical medicine. RECENT FINDINGS: Since the landmark discovery that bone morphogenetic protein receptor type II (BMPR2) mutations cause the majority of cases of familial PAH, investigators have discovered mutations in genes that cause PAH in families without BMPR2 mutations, including the type I receptor ACVRL1 and the type III receptor ENG (both associated with hereditary hemorrhagic telangiectasia), caveolin-1 (CAV1), and a gene (KCNK3) encoding a two-pore potassium channel. Mutations in these genes cause an autosomal-dominant predisposition to PAH in which a fraction of mutation carriers develop PAH (incomplete penetrance). In 2014, scientists discovered mutations in eukaryotic initiation factor 2 alpha kinase 4 (EIF2AK4) that cause pulmonary capillary hemangiomatosis and pulmonary veno-occlusive disease, an autosomal recessively inherited disorder. SUMMARY: The discovery that some forms of pulmonary hypertension are heritable and can be genetically defined adds important opportunities for physicians to educate their patients and their families to understand the potential risks and benefits of genetic testing.

Noncontinuously binding loop-out primers for avoiding problematic DNA sequences in PCR and sanger sequencing.

We present a method in which noncontinuously binding (loop-out) primers are used to exclude regions of DNA that typically interfere with PCR amplification and/or analysis by Sanger sequencing. Several scenarios were tested using this design principle, including M13-tagged PCR primers, non-M13-tagged PCR primers, and sequencing primers. With this technique, a single oligonucleotide is designed in two segments that flank, but do not include, a short region of problematic DNA sequence. During PCR amplification or sequencing, the problematic region is looped-out from the primer binding site, where it does not interfere with the reaction. Using this method, we successfully excluded regions of up to 46 nucleotides. Loop-out primers were longer than traditional primers (27 to 40 nucleotides) and had higher melting temperatures. This method allows the use of a standardized PCR protocol throughout an assay, keeps the number of PCRs to a minimum, reduces the chance for laboratory error, and, above all, does not interrupt the clinical laboratory workflow.

EIF2AK4 mutations in pulmonary capillary hemangiomatosis.

BACKGROUND: Pulmonary capillary hemangiomatosis (PCH) is a rare disease of capillary proliferation of unknown cause and with a high mortality. Families with multiple affected individuals with PCH suggest a heritable cause although the genetic etiology remains unknown. METHODS: We used exome sequencing to identify a candidate gene for PCH in a family with two affected brothers. We then screened 11 unrelated patients with familial (n = 1) or sporadic (n = 10) PCH for mutations. RESULTS: Using exome sequencing, we identified compound mutations in eukaryotic translation initiation factor 2 α kinase 4 (EIF2AK4) (formerly known as GCN2) in both affected brothers. Both parents and an unaffected sister were heterozygous carriers. In addition, we identified two EIF2AK4 mutations in each of two of 10 unrelated individuals with sporadic PCH. EIF2AK4 belongs to a family of kinases that regulate angiogenesis in response to cellular stress. CONCLUSIONS: Mutations in EIF2AK4 are likely to cause autosomal-recessive PCH in familial and some nonfamilial cases.

Cystic fibrosis testing in a referral laboratory: results and lessons from a six-year period.

BACKGROUND: The recent introduction of high throughput sequencing technologies into clinical genetics has made it practical to simultaneously sequence many genes. In contrast, previous technologies limited sequencing based tests to only a handful of genes. While the ability to more accurately diagnose inherited diseases is a great benefit it introduces specific challenges. Interpretation of missense mutations continues to be challenging and the number of variants of uncertain significance continues to grow. RESULTS: We leveraged the data available at ARUP Laboratories, a major reference laboratory, for the CFTR gene to explore specific challenges related to variant interpretation, including a focus on understanding ethnic-specific variants and an evaluation of existing databases for clinical interpretation of variants. In this study we analyzed 555 patients representing eight different ethnic groups. We observed 184 different variants, most of which were ethnic group specific. Eighty-five percent of these variants were present in the Cystic Fibrosis Mutation Database, whereas the Human Mutation Database and dbSNP/1000 Genomes had far fewer of the observed variants. Finally, 21 of the variants were novel and we report these variants and their clinical classifications. CONCLUSIONS: Based on our analyses of data from six years of CFTR testing at ARUP Laboratories a more comprehensive, clinical grade database is needed for the accurate interpretation of observed variants. Furthermore, there is a particular need for more and better information regarding variants from individuals of non-Caucasian ethnicity.