Patterns of mosaicism for sequence and copy-number variants discovered through clinical deep sequencing of disease-related genes in one million individuals.

DNA variants that arise after conception can show mosaicism, varying in presence and extent among tissues. Mosaic variants have been reported in Mendelian diseases, but further investigation is necessary to broadly understand their incidence, transmission, and clinical impact. A mosaic pathogenic variant in a disease-related gene may cause an atypical phenotype in terms of severity, clinical features, or timing of disease onset. Using high-depth sequencing, we studied results from one million unrelated individuals referred for genetic testing for almost 1,900 disease-related genes. We observed 5,939 mosaic sequence or intragenic copy number variants distributed across 509 genes in nearly 5,700 individuals, constituting approximately 2% of molecular diagnoses in the cohort. Cancer-related genes had the most mosaic variants and showed age-specific enrichment, in part reflecting clonal hematopoiesis in older individuals. We also observed many mosaic variants in genes related to early-onset conditions. Additional mosaic variants were observed in genes analyzed for reproductive carrier screening or associated with dominant disorders with low penetrance, posing challenges for interpreting their clinical significance. When we controlled for the potential involvement of clonal hematopoiesis, most mosaic variants were enriched in younger individuals and were present at higher levels than in older individuals. Furthermore, individuals with mosaicism showed later disease onset or milder phenotypes than individuals with non-mosaic variants in the same genes. Collectively, the large compendium of variants, disease correlations, and age-specific results identified in this study expand our understanding of the implications of mosaic DNA variation for diagnosis and genetic counseling.

Optimising Access to Healthcare for Patients Experiencing Homelessness in Hospital Emergency Departments.

The ED is often the first and sometimes the only place where people experiencing homelessness seek medical assistance. While access to primary healthcare is a preferable and more cost-effective alternative to ED, for many reasons, people experiencing homelessness are much less likely to have a regular General Practitioner compared to those living in stable accommodation. Drawing on a growing body of emergency care and homelessness literature and practice, we have synthesised four potential interventions to optimise access to care when people experiencing homelessness present to an ED. Although EDs are in no way responsible for resolving the complex health and social issues of their local homeless population, they are a common contact point and therefore present an opportunity to improve access to healthcare.

Improving Emergency Access to Human Immunodeficiency Virus Prophylaxis for Patients Evaluated After Sexual Assault.

INTRODUCTION: Patients evaluated after sexual assault may benefit from nonoccupational postexposure prophylaxis (nPEP) to prevent infection with HIV, yet multiple barriers may prohibit nPEP delivery. The IN-STEP (Integrating nPEP after Sexual Trauma in Emergency Practice) project was designed to improve access to HIV screening and prevention for patients evaluated in the emergency department (ED) of our academic hospital after a sexual assault. METHODS: The IN-STEP team identified and addressed four key areas for improvement: (1) training of ED providers to perform nPEP assessments; (2) access to HIV testing in the ED; (3) provision of nPEP medications, using a patient-centered approach; and (4) continuity of care between the ED and follow-up sites in the community. Improvements were implemented using parallel plan-do-study-act cycles corresponding to these four key areas. RESULTS: IN-STEP resulted in significant systems improvements in HIV screening, prevention, and continuity of care. This program not only improved the care of patients affected by sexual assault but also those evaluated for HIV due to other indications. CONCLUSIONS: Involvement of a multidisciplinary leadership team, clear delineation of a patient-centered project focus, and coordination across four parallel areas for improvement were useful for completing this complex effort.

A computational study of mental health awareness campaigns on social media.

As public discourse continues to progress online, it is important for mental health advocates, public health officials, and other curious parties and stakeholders, ranging from researchers, to those affected by the issue, to be aware of the advancing new mediums in which the public can share content ranging from useful resources and self-help tips to personal struggles with respect to both illness and its stigmatization. A better understanding of this new public discourse on mental health, often framed as social media campaigns, can help perpetuate the allocation of sparse mental health resources, the need for educational awareness, and the usefulness of community, with an opportunity to reach those seeking help at the right moment. The objective of this study was to understand the nature of and engagement around mental health content shared on mental health campaigns, specifically #MyTipsForMentalHealth on Twitter around World Mental Health Awareness Day in 2017. We collected 14,217 Twitter posts from 10,805 unique users between September and October 2017 that contained the hashtag #MyTipsForMentalHealth. With the involvement of domain experts, we hand-labeled 700 posts and categorized them as (a) Fact, (b) Stigmatizing, (c) Inspirational, (d) Medical/Clinical Tip, (e) Resource Related, (f) Lifestyle or Social Tip or Personal View, and (g) Off Topic. After creating a "seed" machine learning classifier, we used both unsupervised and semi supervised methods to classify posts into the various expert identified topical categories. We also performed a content analysis to understand how information on different topics spread through social networks. Our support vector machine classification algorithm achieved a mean cross-validation accuracy of 0.81 and accuracy of 0.64 on unseen data. We found that inspirational Twitter posts were the most spread with a mean of 4.17 retweets, and stigmatizing content was second with a mean of 3.66 retweets. Classification of social media-related mental health interactions offers valuable insights on public sentiment as well as a window into the evolving world of online self-help and the varied resources within. Our results suggest an important role for social media-based peer support to not only guide information seekers to useful content and local resources but also illuminate the socially-insular aspects of stigmatization. However, our results also reflect the challenges of quantifying the heterogeneity of mental health content on social media and the need for novel machine learning methods customized to the challenges of the field.

The Use of Baclofen as a Treatment for Alcohol Use Disorder: A Clinical Practice Perspective.

Alcohol use disorder (AUD) is a brain disorder associated with high rates of mortality and morbidity worldwide. Baclofen, a selective gamma-aminobutyric acid-B (GABA-B) receptor agonist, has emerged as a promising drug for AUD. The use of this drug remains controversial, in part due to uncertainty regarding dosing and efficacy, alongside concerns about safety. To date there have been 15 randomized controlled trials (RCTs) investigating the use of baclofen in AUD; three using doses over 100 mg/day. Two additional RCTs have been completed but have not yet been published. Most trials used fixed dosing of 30-80 mg/day. The other approach involved titration until the desired clinical effect was achieved, or unwanted effects emerged. The maintenance dose varies widely from 30 to more than 300 mg/day. Baclofen may be particularly advantageous in those with liver disease, due to its limited hepatic metabolism and safe profile in this population. Patients should be informed that the use of baclofen for AUD is as an "off-label" prescription, that no optimal fixed dose has been established, and that existing clinical evidence on efficacy is inconsistent. Baclofen therapy requires careful medical monitoring due to safety considerations, particularly at higher doses and in those with comorbid physical and/or psychiatric conditions. Baclofen is mostly used in some European countries and Australia, and in particular, for patients who have not benefitted from the currently used and approved medications for AUD.

Tackling Health Disparities for People Who Are Homeless? Start with Social Determinants.

BACKGROUND: Homelessness is associated with enormous health inequalities, including shorter life expectancy, higher morbidity and greater usage of acute hospital services. Viewed through the lens of social determinants, homelessness is a key driver of poor health, but homelessness itself results from accumulated adverse social and economic conditions. Indeed, in people who are homeless, the social determinants of homelessness and health inequities are often intertwined, and long term homelessness further exacerbates poor health. Aggregated health service data can mask this, and case histories thus provide important insights. METHODS: This paper presents three case histories of homeless patients seen at an inner city public hospital in Perth, Western Australia. The case histories draw on several data sources: hospital data, information collected from rough sleepers and clinical observations. Estimates of the cost to the health system of the observed hospital usage by the three patients are included. FINDINGS: The case histories illustrate the interplay of social determinants of health in homelessness that help explain the high level of hospital usage by rough sleepers. The cumulative healthcare costs for the three individuals over a 33 months period were substantial. Hospital attendance plummeted even in the short term when housing needs were addressed. CONCLUSIONS: Treating homelessness as a combined health and social issue is critical to improving the abysmal health outcomes of people experiencing homelessness. In addition, the enormous economic costs of hospital care for people who are homeless can be reduced when housing and other social determinants are taken into account.

Assessing copy number from exome sequencing and exome array CGH based on CNV spectrum in a large clinical cohort.

PURPOSE: Detection of copy-number variation (CNV) is important for investigating many genetic disorders. Testing a large clinical cohort by array comparative genomic hybridization provides a deep perspective on the spectrum of pathogenic CNV. In this context, we describe a bioinformatics approach to extract CNV information from whole-exome sequencing and demonstrate its utility in clinical testing. METHODS: Exon-focused arrays and whole-genome chromosomal microarray analysis were used to test 14,228 and 14,000 individuals, respectively. Based on these results, we developed an algorithm to detect deletions/duplications in whole-exome sequencing data and a novel whole-exome array. RESULTS: In the exon array cohort, we observed a positive detection rate of 2.4% (25 duplications, 318 deletions), of which 39% involved one or two exons. Chromosomal microarray analysis identified 3,345 CNVs affecting single genes (18%). We demonstrate that our whole-exome sequencing algorithm resolves CNVs of three or more exons. CONCLUSION: These results demonstrate the clinical utility of single-exon resolution in CNV assays. Our whole-exome sequencing algorithm approaches this resolution but is complemented by a whole-exome array to unambiguously identify intragenic CNVs and single-exon changes. These data illustrate the next advancements in CNV analysis through whole-exome sequencing and whole-exome array.Genet Med 17 8, 623-629.

Tandem duplication PCR: an ultrasensitive assay for the detection of internal tandem duplications of the FLT3 gene.

Internal tandem duplication (ITD) mutations of the FLT3 gene have been associated with a poor prognosis in acute myeloid leukemia. Detection of ITD-positive minor clones at the initial diagnosis and during the minimal residual disease stage may be essential. We previously designed a delta-PCR strategy to improve the sensitivity to 0.1% ITD-positive leukemia cells and showed that minor mutants with an allele burden of <1% can be clinically significant. In this study, we report on tandem duplication PCR (TD-PCR), a modified inverse PCR assay, and demonstrate a limit of detection of a few molecules of ITD mutants. The TD-PCR was initially designed to confirm ITD mutation of an amplicon, which was undetectable by capillary electrophoresis and was incidentally isolated by a molecular fraction collecting tool. Subsequently, TD-PCR detected ITD mutation in 2 of 77 patients previously reported as negative for ITD mutation by a standard PCR assay. TD-PCR can also potentially be applied to monitor minimal residual disease with high analytic sensitivity in a portion of ITD-positive acute myeloid leukemia patients. Further studies using TD-PCR to detect ITD mutants at diagnosis may clarify the clinical significance of those ITD mutants with extremely low allele burden.

Exon-level array CGH in a large clinical cohort demonstrates increased sensitivity of diagnostic testing for Mendelian disorders.

PURPOSE: Mendelian disorders are most commonly caused by mutations identifiable by DNA sequencing. Exonic deletions and duplications can go undetected by sequencing, and their frequency in most Mendelian disorders is unknown. METHODS: We designed an array comparative genomic hybridization (CGH) test with probes in exonic regions of 589 genes. Targeted testing was performed for 219 genes in 3,018 patients. We demonstrate for the first time the utility of exon-level array CGH in a large clinical cohort by testing for 136 autosomal dominant, 53 autosomal recessive, and 30 X-linked disorders. RESULTS: Overall, 98 deletions and two duplications were identified in 53 genes, corresponding to a detection rate of 3.3%. Approximately 40% of positive findings were deletions of only one or two exons. A high frequency of deletions was observed for several autosomal dominant disorders, with a detection rate of 2.9%. For autosomal recessive disorders, array CGH was usually performed after a single mutation was identified by sequencing. Among 138 individuals tested for recessive disorders, 10.1% had intragenic deletions. For X-linked disorders, 3.5% of 313 patients carried a deletion or duplication. CONCLUSION: Our results demonstrate that exon-level array CGH provides a robust option for intragenic copy number analysis and should routinely supplement sequence analysis for Mendelian disorders.

A virtual pyrogram generator to resolve complex pyrosequencing results.

We report a freely available software program, Pyromaker, which generates simulated traces for pyrosequencing results based on user inputs. Simulated pyrograms can aid in the analysis of complex pyrosequencing results in which various hypothesized mutations can be tested, and the resultant pyrograms can be matched with the actual pyrogram. We validated the software using the actual pyrograms for common KRAS gene mutations as well as several mutations in the BRAF, GNAS, and p53 genes. We demonstrate that all 18 possible single-base mutations within codons 12 and 13 of KRAS generate unique pyrosequencing traces and highlight the distinctions between them. We further show that all reported codon 12 and 13 complex mutations produce unique pyrograms. However, some complex mutations are indistinguishable from single-base mutations. For complicated pyrograms, Pyromaker was used in two modes, one in which hypothesis-based simulated pyrograms were pattern-matched with the actual pyrograms. In a second strategy with only the pyrogram, Pyromaker was used to identify the underlying mutation by iteratively reconstructing the mutant pyrogram. Either strategy was able to successfully identify the complex mutations, which were confirmed by cloning and sequencing. Using two examples of KRAS codon 12 mutations (specifically GGT→TTT, G12F and GGT→GAG, G12E), we report which combinations of five approaches permit unambiguous mutation identification. The most efficient approach was found to be pyrosequencing with Pyromaker.