Adopting duplex sequencing technology for genetic toxicity testing: A proof-of-concept mutagenesis experiment with N-ethyl-N-nitrosourea (ENU)-exposed rats.

Duplex sequencing (DS) is an error-corrected next-generation sequencing method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors in consensus sequences. The resulting background of less than one artifactual mutation per 107 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DS-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues⁠, a considerable advancement compared to currently used in vivo gene mutation assays.

Error-corrected duplex sequencing enables direct detection and quantification of mutations in human TK6 cells with strong inter-laboratory consistency.

Error-corrected duplex sequencing (DS) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DS to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25-200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DS mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A significant increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (r = 0.97). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The consistent results across the three time points suggest that 48 h may be sufficient for mutation analysis post-exposure. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DS for characterizing chemical mutagenicity in both research and regulatory evaluation.

Adopting Duplex Sequencing™ Technology for Genetic Toxicity Testing: A Proof-of-Concept Mutagenesis Experiment with N-Ethyl-N-Nitrosourea (ENU)-Exposed Rats.

Duplex sequencing (DuplexSeq) is an error-corrected next-generation sequencing (ecNGS) method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors by comparing grouped strand sequencing reads. The resulting background of less than one artifactual mutation per 10 7 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DuplexSeq-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues, a considerable advancement compared to currently used in vivo gene mutation assays. HIGHLIGHTS: DuplexSeq is an ultra-accurate NGS technology that directly quantifies mutationsENU-dependent mutagenesis was detected 24 h post-exposure in proliferative tissuesMultiple tissues exhibited the canonical ENU mutation spectrum 7 d after exposureResults obtained with DuplexSeq were highly concordant between laboratoriesThe Rat-50 Mutagenesis Assay is promising for applications in genetic toxicology.

Development and Refinement of the American Occupational Therapy Association's Knowledge Translation Toolkit.

IMPORTANCE: Occupational therapy practitioners are expected to translate promising discoveries from empirical research into routine practice with their clients. However, complex barriers can influence practitioners' knowledge translation (KT) efforts, leading the American Occupational Therapy Association's Evidence-Based Practice (EBP) group to develop the KT Toolkit tailored to the perceived needs of occupational therapists and occupational therapy assistants. OBJECTIVE: To identify common barriers to implementing EBPs and potential strategies to support EBP uptake. DESIGN: Cross-sectional survey. SETTING: United States. PARTICIPANTS: Occupational therapy practitioners. OUTCOMES AND MEASURES: Data underwent descriptive and directed content analysis, the latter of which was guided by the Consolidated Framework for Implementation Research. RESULTS: Occupational therapy survey respondents (N = 818) identified common EBP implementation barriers (e.g., lack of time and resources, difficulty understanding research findings). Initial KT Toolkit content was developed to address these barriers and included resources for searching for, analyzing, and applying evidence in practice. CONCLUSIONS AND RELEVANCE: Survey findings have informed the development of the KT Toolkit, which includes resources designed to support occupational therapy practitioners' EBP implementation efforts. This KT Toolkit is available at AOTA.org and will be continuously revised and updated on an ongoing basis. What This Article Adds: Several barriers limit the extent to which occupational therapy practitioners can implement evidence with their client populations. The KT Toolkit is directly informed by practitioner input and provides resources to support practitioners in their efforts to translate knowledge into real-world practice.

Healthcare providers' role in providing sexual and reproductive health information to people with intellectual and developmental disabilities: A qualitative study.

BACKGROUND: Individuals with intellectual and developmental disabilities demonstrate disparities in sexual and reproductive health (SRH) compared to individuals without disabilities (e.g., lack of sexual education and knowledge, increased rates of abuse, unplanned pregnancies and sexually transmitted infections). Therefore, the purpose of this study was to identify topics healthcare providers address and perceived barriers and supports to SRH education. METHODS: We conducted semi-structured interviews with healthcare providers (N = 12). RESULTS: Providers address relationships, safety, protection and appropriate sexual behaviours with clients with intellectual and developmental disabilities. Parent education and client-centred care were identified as supports, while the patient's level of understanding, the provider's lack of knowledge or access to resources and to appropriate referrals were identified as barriers to SRH education. CONCLUSION: Future studies are needed to link providers to resources they can use to provide comprehensive, accessible SRH education for clients with intellectual and developmental disabilities.

Sex Education Practices for People With Intellectual and Developmental Disabilities: A Qualitative Study.

IMPORTANCE: People with intellectual and developmental disabilities (IDD) express a clear interest in intimate relationships but face many barriers to receiving sex education (SE) that would support their engagement in these relationships. OBJECTIVE: To understand barriers to, the context of, and recommendations for SE for people with IDD. DESIGN: Qualitative study design with interviews and focus groups with four key stakeholder groups. Data were analyzed using a constant comparative approach. PARTICIPANTS: Participants were 8 youths with IDD, 9 parents, 12 health care providers, and 8 educators. RESULTS: Four barriers to SE were identified: (1) values and cultural issues, (2) parental attitudes toward their child's sexuality, (3) a lack of organizational policies and standards, and (4) limited professional education or societal biases. These barriers contribute to a SE context primarily initiated by people with IDD or provided reactively. The participants recommended proactive, formal SE provided by multiple stakeholders throughout adulthood. CONCLUSIONS AND RELEVANCE: Stakeholders should advocate for policies, standards, and additional training for parents, educators, and health care providers to support SE for people with IDD throughout adulthood. What This Article Adds: Barriers to SE contribute to the current context in which SE is shared with people with IDD. Stakeholders can advocate for policies, standards, and training to overcome these barriers and support recommendations for proactive, formal SE provided by multiple stakeholders through adulthood.

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.

Genetic toxicity testing using human in vitro organotypic airway cultures: Assessing DNA damage with the CometChip and mutagenesis by Duplex Sequencing.

The organotypic human air-liquid-interface (ALI) airway tissue model has been used as an in vitro cell culture system for evaluating the toxicity of inhaled substances. ALI airway cultures are highly differentiated, which has made it challenging to evaluate genetic toxicology endpoints. In the current study, we assayed DNA damage with the high-throughput CometChip assay and quantified mutagenesis with Duplex Sequencing, an error-corrected next-generation sequencing method capable of detecting a single mutation per 107 base pairs. Fully differentiated human ALI airway cultures were treated from the basolateral side with 6.25 to 100 µg/mL ethyl methanesulfonate (EMS) over a period of 28 days. CometChip assays were conducted after 3 and 28 days of treatment, and Duplex Sequencing after 28 days of treatment. Treating the airway cultures with EMS resulted in time- and concentration-dependent increases in DNA damage and a concentration-dependent increase in mutant frequency. The mutations observed in the EMS-treated cultures were predominantly C → T transitions and exhibited a unique trinucleotide signature relative to the negative control. Measurement of physiological endpoints indicated that the EMS treatments had no effect on anti-p63-positive basal cell frequency, but produced concentration-responsive increases in cytotoxicity and perturbations in cell morphology, along with concentration-responsive decreases in culture viability, goblet cell and anti-Ki67-positive proliferating cell frequency, cilia beating frequency, and mucin secretion. The results indicate that a unified 28-day study can be used to measure several important safety endpoints in physiologically relevant human in vitro ALI airway cultures, including DNA damage, mutagenicity, and tissue-specific general toxicity.

Impact of COVID-19 on services for people with disabilities and chronic health conditions.

BACKGROUND: People with disabilities and chronic health conditions rely on a range of services and supports to complete daily tasks, maintain health, and participate in the community. Preliminary research suggests the COVID-19 pandemic greatly disrupted these services and this population may be particularly susceptible to unemployment. OBJECTIVE: Describe employment and service disruptions for individuals with disabilities and chronic health conditions during the onset of community-based spread of COVID-19 in the United States. METHODS: Adults with disabilities and chronic health conditions completed online surveys to report employment and service changes via multiple choice and open-ended questions. Multiple choice questions were analyzed using descriptive statistics; open-ended responses were coded using content analysis. RESULTS: Participants (n = 109): 79.8% female, 88.1% white, 77.121% completed a 4-year college degree or greater, 61.4% had annual income ≥$45,000. Only 14.9% of survey respondents reported disruptions in employment. On average, 54.0% of service changes were due to discontinuation, including loss of physical therapy, job coaching, community organizations, transportation, and peer supports. Other changes included a shift to virtual service delivery and family members taking the role of service providers. CONCLUSIONS: Individuals with chronic health conditions and disabilities experienced service disruptions, even in a sample with considerably more economic, social, and educational privilege than the general population of people with chronic health conditions and disabilities in the United States.

Assessing Activity of Daily Living Task Performance Among Autistic Adults.

Activities of daily living (ADLs) are activities that people engage in on a routine basis, such as brushing their teeth, preparing a meal, and caring for their child. Independence with ADLs is associated with better outcomes in independent living, education, employment, relationships, and mental health. Therefore, this perspective piece includes a review of the literature and assessment databases to identify and summarize ADL assessments for Autistic adults. These assessments were compared and analyzed using the neurodiversity paradigm. Specifically, we compared assessments using predetermined priorities the authors identified: (1) assessment type, (2) inclusivity, and (3) performance factors. We identified five unique norm-referenced measures, four performance-based measures, and a variety of checklists, surveys, questionnaires, and/or interviews used to assess ADL performance among Autistic adults. The authors present their perspectives regarding the challenges with the current assessments, including the high-frequency use of norm-referenced assessments, lack of inclusivity, and failure to consider performance factors (e.g., sensory, motor, and emotional), and the paucity of assessments designed specifically for Autistic adults older than 30 years. In response to these challenges, we recommend researchers partner with Autistic adults to develop a new assessment tool. If researchers or clinicians are using existing measures, we recommend that they utilize self-report over proxy-report and include methods to improve the accessibility of the assessment. We also recommend that clinicians and researchers offer breaks, comfort objects, or sensory modifications during the assessment to decrease anxiety; and ask follow-up questions to understand whether environment or emotional health are impacting one's ADL performance. Lay summary: Why is this topic important?: Activity of daily living (ADL) assessments are used to determine what Autistics can and cannot do in their day-to-day life, what services they may be eligible for, and to monitor gains. However, we struggled to find an assessment that was useful and relevant from an Autistic point-of-view.What is the purpose of this article?: The purpose of this article was to review and evaluate current ADL assessments usefulness for Autistics and provide recommendations for improving the ADL assessment process.What is the perspective of the authors?: The first author is an Autistic social worker and the second author is an occupational therapist and postdoctoral fellow. The authors' perspective is based in the neurodiversity paradigm and social model of disability, which centers on respecting and acknowledging differences in the brain and their effects on Autistics' lives. We believe in strengths-based approaches versus deficit-based models.What did you find about this topic?: We found 17 measurement tools, some that compared Autistic ADL performance with neurotypical performance, a few that were observation-based meaning the researcher or clinician watched the Autistic person complete the ADLs, and many forms or guides that asked questions about ADL performance. Only six measures allowed Autistic people to respond to the questions themselves, whereas the rest of the measures had someone else respond for them. These measures did not include questions about how sensory differences (e.g., feeling upset by the feeling of jeans or the taste of minty toothpaste) or feeling sad or nervous may also impact ADL performance.What do the authors recommend?: We recommend that researchers partner with Autistic adults to make new ADL assessments. If researchers or clinicians are using previously made ADL assessments, we recommend that they use self-report and adapt the materials to make it easier to understand (e.g., using pictures). We also recommend that researchers and clinicians ask Autistics what they need or want to make the assessment easier and more comfortable for them. Finally, researchers and clinicians should ask follow-up questions about sensory differences and whether someone is feeling sad or nervous to know how this impacts their ability to do their ADL tasks.How will these recommendations help autistic adults now or in the future?: These recommendations will help Autistic adults be more involved in the evaluation process, which will make the assessments more trustworthy and relevant to Autistics. This also could help more Autistic people get services and supports that are useful to them. Finally, this may help researchers when monitoring if these supports or services actually work.

Scoping Review of Self-Advocacy Education Interventions to Improve Care.

Self-advocacy is a client's ability to represent one's interests when managing disease or disability. Self-advocacy may increase one's ability to seek, evaluate, and use information to promote health, yet little is known about the role of occupational therapy in promoting self-advocacy. This scoping review aims to identify interventions within occupational therapists' scope of practice to improve self-advocacy. A literature search was conducted through Academic Search Complete to identify interventions within the scope of occupational therapy that promote self-advocacy. All levels of evidence were included, and articles were reviewed for inclusion by two authors. Included articles were charted for level of evidence, objectives, participants, and results. Fourteen articles met the criteria. Interventions included interactive multimedia interventions, peer-led educational groups, writing interventions, job counseling and advocacy, and disease-specific advocacy programs. Occupational therapists are well-equipped to intervene and promote self-advocacy through workplace modification, utilizing assistive technologies, and facilitating peer-led educational groups.

Mitochondrially-targeted APOBEC1 is a potent mtDNA mutator affecting mitochondrial function and organismal fitness in Drosophila.

Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In Drosophila, knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. Here we describe a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. mito-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and mito-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make mito-APOBEC1 an excellent mtDNA mutator model for ageing research.

Sexually transmitted infections in privately insured adults with intellectual and developmental disabilities.

Aim: Individuals with intellectual and developmental disabilities (I/DD) may have an increased risk of sexually transmitted infections (STIs) due to limited sexual health education and higher rates of sexual abuse, yet little is known about the prevalence of STIs and STI testing in this population. Methods: This study compared national samples of privately insured individuals with (n = 25,193) and without I/DD (n = 25,193) on the prevalence of STIs and STI testing. Results: In multivariable models, individuals with I/DD were significantly less likely to have an STI diagnosis and no difference was found between groups on the odds of STI testing overall. Conclusion: Findings may, in part, be explained by fewer sexual experiences, increased supervision in social settings and delayed onset of sexual activity among those with I/DD.

Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS).

Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.