Genomic signature of Fanconi anaemia DNA repair pathway deficiency in cancer.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

Author Correction: Determination of RNA structural diversity and its role in HIV-1 RNA splicing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Determination of RNA structural diversity and its role in HIV-1 RNA splicing.

Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors1,2. Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements3. Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name 'detection of RNA folding ensembles using expectation-maximization' (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages4, our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized5 alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis6-8 that heterogeneity in RNA conformation regulates splice-site use and viral gene expression.

Integrated genome and transcriptome analyses reveal the mechanism of genome instability in ataxia with oculomotor apraxia 2.

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.

Delayed DNA replication in haploid human embryonic stem cells.

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.

Impact of regional differences and neighborhood socioeconomic deprivation on the outcomes of patients with lower extremity wounds evaluated by a limb-preservation service.

OBJECTIVE: Management of lower extremity (LE) wounds has evolved with the establishment of specialized limb preservation services. Although clinical factors contribute to limb outcomes, socioeconomic status and community factors also influence the risk for limb loss. The Distressed Community Index (DCI) score is a validated index of social deprivation created to provide an objective measure of economic well-being in United States communities. Few studies have examined the influence of geographic deprivation on outcomes in patients with LE wounds. We examined relationships between socioeconomic deprivation and outcomes of inpatients evaluated by a dedicated limb preservation service (Functional Limb Extremity Service [FLEX]). METHODS: Inpatients referred to FLEX over a 5-year period were included. Wound, Ischemia, foot Infection (WIfI) staging was collected. DCI scores were determined using seven indices based on ZIP Code. Outcomes included any minor or major amputations, any endovascular or open LE revascularization, or wound care procedures. Disease etiology, demographic, and anthropometric data were collected. Associations between neighborhood deprivation and limb-specific outcomes were evaluated in models for the DCI and each of its components separately. RESULTS: A total of 677 patients were included. Thirty-eight percent were female, with a mean age of 64 years. Sixty percent had WIfI stage 3 or 4 risk of amputation, and 43% had WIfI stage 3 or 4 risk of revascularization. Mean ankle-brachial index and toe pressure were 0.96 (standard deviation [SD], 0.43) and 80 (SD, 57) mmHg. Thirty-five percent were non-White. Amputation was performed in 31% of patients, whereas 17% underwent revascularization. The mean distress score was 64 (SD, 24). Mean DCI scores did not differ across WIfI scores. Likewise, overall DCI distress score was not related to any of the outcomes in univariable or multivariable linear regression models. In univariable linear regression models for amputation, higher poverty rate (odds ratio for SD increase 1.20; 95% confidence interval, 1.02-1.42; P = .025) was significantly associated with the outcome. In multivariable models, neither DCI distress score nor any of its components remained significantly associated with the outcome. CONCLUSIONS: Despite known racial disparities in limb-specific outcomes, an aggregate measure of community level distress was not found to be related to outcomes. Although the poverty rate demonstrated a significant relationship with amputation in univariable analysis, this association was not found in multivariable models. Notably, non-White race emerged as a predictor of amputation, underscoring the importance of addressing racial disparities in LE outcomes. Further investigation of potential determinants of LE outcomes is needed, particularly the interaction of such factors with race.

Contemporary outcomes for arterial reconstruction with non-saphenous vein cryo-preserved conduits.

OBJECTIVE: Cryopreserved (CP) products are utilized during challenging cases when autogenous or prosthetic conduit use is not feasible. Despite decades of experience with cadaveric greater saphenous vein (GSV), there is limited available data regarding the outcomes and patency of other CP products, specifically arterial and deep venous grafts. This study was designed to evaluate outcomes of non-GSV CP conduits in patients undergoing urgent, emergent, and elective arterial reconstruction at our institution. We hypothesized that non-GSV CP allografts have adequate patency and outcomes and are therefore a feasible alternative to GSV in settings where autologous graft is unavailable or prosthetic grafts are contraindicated. METHODS: This study was approved by the Institutional Review Board at our institution. We retrospectively reviewed charts of patients undergoing arterial reconstructions using CP conduits from 2010 to 2022. Data collected included demographics, comorbidities, smoking status, indications for surgery, indication for CP conduit use, anatomic reconstruction, urgency of procedure, and blood loss. Time-to-event outcomes included primary and secondary graft patency rates, follow-up amputations, and mortality; other complications included follow-up infection/reinfection and 30-day complications, including return to the operating room and perioperative mortality. Time-to-event analyses were evaluated using product-limit survival estimates. RESULTS: Of 96 identified patients receiving CP conduits, 56 patients received non-GSV conduits for 66 arterial reconstructions. The most common type of non-GSV CP product used was femoral artery (31 patients), followed by aorto-iliac artery (22 patients), and femoral vein (19 patients), with some patients receiving more than one reconstruction or CP product. Patients were mostly male (75%), with a mean age of 63.1 years and a mean body mass index of 26.7 kg/m2. Indications for CP conduit use included infection in 53 patients, hostile environment in 36 patients, contaminated field in 30 patients, tissue coverage concerns in 30 patients, inadequate conduit in nine patients, and patient preference in one patient. Notably, multiple patients had more than one indication. Most surgeries (95%) were performed in urgent or emergent settings. Supra-inguinal reconstructions were most common (53%), followed by extra-anatomic bypasses (47%). Thirty-day mortality occurred in 10 patients (19%). Fifteen patients (27%) required return to the operating room for indications related to the vascular reconstructions, with 10 (18%) cases being unplanned and five (9%) cases planned/staged. Overall survival at 6, 12, and 24 months was 80%, 68%, and 59%, respectively. Primary patency at 6, 12, and 24 months was 86%, 70%, and 62%, respectively. Amputation freedom at 6 months, 12 months, and 24 months was 98%, 95%, and 86%, respectively for non-traumatic indications. CONCLUSIONS: Non-GSV CP products may be used in complex arterial reconstructions when autogenous or prosthetic options are not feasible or available.

EED related overgrowth: First report of multiple members in a single family.

EED is a core component of polycomb repressive complex 2 (PRC2) with EZH2 and SUZ12. PRC2 has H3K27 methyltransferase activity (HMTase) that catalyzes the addition of up to three methyl groups on histone 3 at lysine residue 27 (H3K27). Germline heterozygous variants in EED, SUZ12, and EZH2 have been identified in patients with overgrowth and multiple dysmorphic features. The clinical manifestations of these syndromes significantly overlap: generalized overgrowth, intellectual disability, and scoliosis. To date, 11 unrelated patients have been published with missense variants in EED at highly conserved amino acids. We report three affected members in a family with a previously reported missense variant. All three affected members manifested very similarly, and this represents a homogenous clinical phenotype associated with EED related intellectual disability and overgrowth. This disorder is appropriately called Cohen-Gibson syndrome.

Comparison of predictive accuracy of knee height equations in children.

Knee height can be a proxy for height when standing height cannot be reliably measured. We compared two commonly used equations (Chumlea and Rumapea) that estimate standing height from knee height. We prospectively enrolled 210 children without scoliosis or kyphosis aged 7-12 years (mean age: 10.2 years, 47.6% males) and measured their knee heights and standing heights. A two-tailed T-test was used to compare predicted heights from each of the equations to actual standing height. Chumlea equation was found to be unreliable (p = 0.0376) while Rumapea equation was found to be reliable in estimating standing height (p = 0.878). Additionally, Rumapea equation was also found to be more accurate than Chumlea equation when results were segregated based on gender and race. In conclusion, the Rumapea equation yields more accurate estimates of standing heights than the Chumlea equation in US children aged 7-12 years.

Neuropathy Screening for Patients with Peripheral Vascular Disease Helps to Identify Those at an Increased Risk of Amputation, Revascularization, and Death.

BACKGROUND: Peripheral neuropathy is associated with amputation risk among patients with diabetes mellitus and chronic limb-threatening ischemia (CLTI). Detection of peripheral neuropathy may help identify those who are at an increased risk, but the predictive ability of the screening tool used in patients with peripheral arterial disease (PAD) needs to be more clearly defined. METHODS: Patients referred to vascular surgery clinic for PAD were recruited from a single center. Exclusion criteria were a documented history of neuropathy or prior lower limb amputation. Screening utilized the Michigan Neuropathy Screening Instrument (MNSI). Scores >2.5 were considered abnormal and scores >4 were considered positive for peripheral neuropathy. Limb-specific outcomes of amputation and revascularization as well as a composite outcome including death were modeled using time to event analysis. RESULTS: 86 patients were recruited. Mean age was 67 ± 10.2 years, 30% were women, 24% were black. Mean ankle-brachial index was 0.74 ± 0.3. PAD symptoms at initial evaluation were claudication in 52% of patients and CLTI in 38% of patients. Neuropathy was present in 20% of the cohort with a significantly higher proportion in diabetics (34% vs. 3%; P = 0.0009). Neuropathy was more common in patients with CLTI compared to claudicants (36% vs. 9%; P = 0.011). Forty patients (47%) reached the composite outcome of amputation, revascularization, or death with a median time to event of 16 months. Abnormal MNSI examination was significantly associated with the increased risk of the composite outcome (hazard ratio = 3.19; P 0.0005). CONCLUSIONS: A significant proportion of patients presenting to vascular specialists for PAD have undiagnosed neuropathy. Patients with PAD and neuropathy have an increased risk of amputation, revascularization, and death. Expanding neuropathy screening in vascular surgery clinic visits may help to identify patients at higher risk.

Implementation strategies to support ultrasound thyroid nodule risk stratification: A systematic review.

BACKGROUND: Ultrasound risk stratification can improve the care of patients with thyroid nodules by providing a structured and systematic approach for the evaluation of thyroid nodule features and thyroid cancer risk. The optimal strategies to support implementation of high quality thyroid nodule risk stratification are unknown. This study seeks to summarise strategies used to support implementation of thyroid nodule ultrasound risk stratification in practice and their effects on implementation and service outcomes. METHODS: This is a systematic review of studies evaluating implementation strategies published between January 2000 and June 2022 that were identified on Ovid MEDLINE, Ovid EMBASE, Ovid Cochrane, Scopus, or Web of Science. Screening of eligible studies, data collection and assessment for risk of bias was completed independently and in duplicate. Implementation strategies and their effects on implementation and service outcomes were evaluated and summarised. RESULTS: We identified 2666 potentially eligible studies of which 8 were included. Most implementation strategies were directed towards radiologists. Common strategies to support the implementation of thyroid nodule risk stratification included: tools to standardise thyroid ultrasound reports, education on thyroid nodule risk stratification and use of templates/forms for reporting, and reminders at the point of care. System based strategies, local consensus or audit were less commonly described. Overall, the use of these strategies supported the implementation process of thyroid nodule risk stratification with variable effects on service outcomes. CONCLUSIONS: Implementation of thyroid nodule risk stratification can be supported by development of standardised reporting templates, education of users on risk stratification and reminders of use at the point of care. Additional studies evaluating the value of implementation strategies in different contexts are urgently needed.

Contemporary outcomes for branch renal artery repair.

BACKGROUND: The purpose of this study was to provide an updated description of demographics, technical details, and clinical outcomes of 101 consecutive branch renal artery repairs in 98 patients using cold perfusion. METHODS: A single-institution, retrospective analysis branch renal artery reconstructions was performed between 1987 and 2019. RESULTS: Patients were predominantly Caucasian (80.6%) women (74.5%) with a mean age of 46.8 ± 15.3 years. The mean preoperative systolic and diastolic blood pressures were 170.4 ± 33.0 mm Hg and 99.2 ± 19.9 mm Hg, respectively, requiring a mean of 1.6 ± 1.1 antihypertensive medications. The estimated glomerular filtration rate was 84.0 ± 25.3 mL/min. Most patients (90.2%) were not diabetic and never smokers (68%). Treated pathology included aneurysm (87.4%) and stenosis (23.3%) with histology demonstrating fibromuscular dysplasia (44.4%), dissection (5.1%), and degenerative not otherwise specified (50.5%). The right renal arteries were most frequently treated (44.2%), with a mean of 3.1 ± 1.5 branches involved. Reconstruction was accomplished using bypass in 90.3% of cases using aortic inflow in 92.7% and a saphenous vein conduit in 92%. Branch vessels served as outflow in 96.9% and syndactylization of branches was used to decrease the number of distal anastomoses in 45.3% of repairs. The mean number of distal anastomoses was 1.5 ± 0.9. Postoperatively, the mean systolic blood pressure improved to 137.9 ± 20.8 mm Hg (mean decrease of 30.5 ± 32.8 mm Hg; P < .0001) and the mean diastolic blood pressure improved to 78.4 ± 12.7 mm Hg (mean decrease of 20.1 ± 20.7 mm Hg; P < .0001) with patients requiring a mean of 1.4 ± 1.0 antihypertensive medications (mean decrease of 0.2 ±1.0 medications; P = .048). The postoperative estimated glomerular filtration rate was 89.1 mL/min (mean increase of 4.1 mL/min; P = .08). The mean length of stay was 9.0 ± 5.8 days and 96.1% of patients were discharged home. The mortality rate was 1% (one patient with liver failure) and the major morbidity rate was 15%. There were five infectious complications (pneumonia, Clostridium difficile, and wound infection) and five patients required return to the operating room (one for nephrectomy, one for bleeding, two for thrombosis, and one for second trimester pregnancy loss requiring dilation and curettage and splenectomy). One patient required temporary dialysis owing to graft thrombosis. Two patients developed arrhythmias. No patients suffered a myocardial infarction, stroke, or limb loss. After 30 days, follow-up data were available for 82 bypasses. At this time, three reconstructions were no longer patent. Intervention was required to retain patency for five bypasses. After 1 year, patency data were available for 61 bypasses and five were no longer patent. Of the five grafts with loss of patency, two underwent intervention in attempt to maintain patency, which subsequently failed. CONCLUSIONS: Repair of renal artery pathology involving the branches can be performed with short- and long-term technical success and significant prospect of decreasing an elevated blood pressure. The operations required to fully address the presenting pathology are often quite complex involving multiple distal anastomoses and consolidation of small secondary branches. The procedure carries a small but significant risk of major morbidity and mortality.

Jansen-de Vries syndrome: Expansion of the PPM1D clinical and phenotypic spectrum in 34 families.

Jansen-de Vries syndrome (JdVS) is a neurodevelopmental condition attributed to pathogenic variants in Exons 5 and 6 of PPM1D. As the full phenotypic spectrum and natural history remain to be defined, we describe a large cohort of children and adults with JdVS. This is a retrospective cohort study of 37 individuals from 34 families with disease-causing variants in PPM1D leading to JdVS. Clinical data were provided by treating physicians and/or families. Of the 37 individuals, 27 were male and 10 female, with median age 8.75 years (range 8 months to 62 years). Four families document autosomal dominant transmission, and 32/34 probands were diagnosed via exome sequencing. The facial gestalt, including a broad forehead and broad mouth with a thin and tented upper lip, was most recognizable between 18 and 48 months of age. Common manifestations included global developmental delay (35/36, 97%), hypotonia (25/34, 74%), short stature (14/33, 42%), constipation (22/31, 71%), and cyclic vomiting (6/35, 17%). Distinctive personality traits include a hypersocial affect (21/31, 68%) and moderate-to-severe anxiety (18/28, 64%). In conclusion, JdVS is a clinically recognizable neurodevelopmental syndrome with a characteristic personality and distinctive facial features. The association of pathogenic variants in PPM1D with cyclic vomiting bears not only medical attention but also further pathogenic and mechanistic evaluation.

Addressing the Increasing Mental Health Distress and Mental Illness Among Young Adults in the United States.

ABSTRACT: Recent surveys show rising numbers of young people who report anxiety and depression. Although much attention has focused on mental health of adolescent youth, less attention has been paid to young people as they transition into adulthood. Multiple factors may have contributed to this steady increase: greater exposure to social media, information, and distressing news via personal electronic devices; increased concerns regarding social determinants of health and climate change; and changing social norms due to increased mental health literacy and reduced stigma. The COVID-19 pandemic may have temporarily exacerbated symptoms and impacted treatment availability. Strategies to mitigate causal factors for depression and anxiety in young adults may include education and skills training for cognitive, behavioral, and social coping strategies, as well as healthier use of technology and social media. Policies must support the availability of health insurance and treatment, and clinicians can adapt interventions to encompass the specific concerns and needs of young adults.

Evidence-Based Assessment of Genes in Dilated Cardiomyopathy.

BACKGROUND: Each of the cardiomyopathies, classically categorized as hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy, has a signature genetic theme. Hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy are largely understood as genetic diseases of sarcomere or desmosome proteins, respectively. In contrast, >250 genes spanning >10 gene ontologies have been implicated in DCM, representing a complex and diverse genetic architecture. To clarify this, a systematic curation of evidence to establish the relationship of genes with DCM was conducted. METHODS: An international panel with clinical and scientific expertise in DCM genetics evaluated evidence supporting monogenic relationships of genes with idiopathic DCM. The panel used the Clinical Genome Resource semiquantitative gene-disease clinical validity classification framework with modifications for DCM genetics to classify genes into categories on the basis of the strength of currently available evidence. Representation of DCM genes on clinically available genetic testing panels was evaluated. RESULTS: Fifty-one genes with human genetic evidence were curated. Twelve genes (23%) from 8 gene ontologies were classified as having definitive (BAG3, DES, FLNC, LMNA, MYH7, PLN, RBM20, SCN5A, TNNC1, TNNT2, TTN) or strong (DSP) evidence. Seven genes (14%; ACTC1, ACTN2, JPH2, NEXN, TNNI3, TPM1, VCL) including 2 additional ontologies were classified as moderate evidence; these genes are likely to emerge as strong or definitive with additional evidence. Of these 19 genes, 6 were similarly classified for hypertrophic cardiomyopathy and 3 for arrhythmogenic right ventricular cardiomyopathy. Of the remaining 32 genes (63%), 25 (49%) had limited evidence, 4 (8%) were disputed, 2 (4%) had no disease relationship, and 1 (2%) was supported by animal model data only. Of the 16 evaluated clinical genetic testing panels, most definitive genes were included, but panels also included numerous genes with minimal human evidence. CONCLUSIONS: In the curation of 51 genes, 19 had high evidence (12 definitive/strong, 7 moderate). It is notable that these 19 genes explain only a minority of cases, leaving the remainder of DCM genetic architecture incompletely addressed. Clinical genetic testing panels include most high-evidence genes; however, genes lacking robust evidence are also commonly included. We recommend that high-evidence DCM genes be used for clinical practice and that caution be exercised in the interpretation of variants in variable-evidence DCM genes.

Functional genomics and gene-environment interaction highlight the complexity of congenital heart disease caused by Notch pathway variants.

Congenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants.

De Novo ZMYND8 variants result in an autosomal dominant neurodevelopmental disorder with cardiac malformations.

PURPOSE: ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. We delineate a novel neurocognitive disorder caused by variants in the ZMYND8 gene. METHODS: An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants. RESULTS: ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the DrosophilaZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function. CONCLUSION: We present genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.

Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants.

PURPOSE: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy). METHODS: A total of 74 families with diverse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases. RESULTS: Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with samples from ≥2 affected individuals or heterozygotes and 10 cases with ≥2 biospecimens. PCR amplicons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing. CONCLUSION: RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long amplicons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data.

Confirmation and variability of the Allee effect inDictyostelium discoideumcell populations, possible role of chemical signaling within cell clusters.

In studies of the unicellular eukaryoteDictyostelium discoideum, many have anecdotally observed that cell dilution below a certain 'threshold density' causes cells to undergo a period of slow growth (lag). However, little is documented about the slow growth phase and the reason for different growth dynamics below and above this threshold density. In this paper, we extend and correct our earlier work to report an extensive set of experiments, including the use of new cell counting technology, that set this slow-to-fast growth transition on a much firmer biological basis. We show that dilution below a certain density (around 104cells ml-1) causes cells to grow slower on average and exhibit a large degree of variability: sometimes a sample does not lag at all, while sometimes it takes many moderate density cell cycle times to recover back to fast growth. We perform conditioned media experiments to demonstrate that a chemical signal mediates this endogenous phenomenon. Finally, we argue that while simple models involving fluid transport of signal molecules or cluster-based signaling explain typical behavior, they do not capture the high degree of variability between samples but nevertheless favor an intra-cluster mechanism.

Prevalence of chronic opioid use in patients with peripheral arterial disease undergoing revascularization.

OBJECTIVE: Opiate use, dependence, and the associated morbidity and mortality are major current public health problems in the United States. Little is known about patterns of opioid use in patients with peripheral arterial disease (PAD). The purpose of this study was to identify the prevalence of chronic preoperative and postoperative prescription opioid use in patients with PAD. A secondary aim was to determine the demographic, comorbid conditions, and operative characteristics associated with chronic opioid use. METHODS: Using a single-institution database of patients with PAD undergoing open or endovascular lower extremity intervention from 2013 to 2014, data regarding opiate use and associated conditions were abstracted for analysis. Patients were excluded if they did not live in North Carolina or surgery was not for PAD. Preoperative (PreCOU) and postoperative chronic opioid use (PostCOU) were defined as consistent opioid prescription filling in the 3 months before and after the index procedure, respectively. Opioid prescription filling was assessed using the North Carolina Controlled Substance Reporting System. Demographics, comorbid conditions, other adjunct pain medication data, and operative characteristics were abstracted from our institutional electronic medical record. Associations with PreCOU were evaluated using the t test, Wilcoxon test, or two-sample median test (continuous), or the χ2 or Fisher exact tests (categorical). RESULTS: A total of 202 patients undergoing open (108; 53.5%) or endovascular (94; 46.5%) revascularization for claudication or critical limb ischemia were identified for analysis. The mean age was 64.6 years, and 36% were female. Claudication was the indication for revascularization in 26.7% of patients, and critical limb ischemia was the indication in 73.3% of patients. The median preoperative ankle-brachial index (ABI) was 0.50. Sixty-eight patients (34%) met the definition for PreCOU. PreCOU was associated with female gender, history of chronic musculoskeletal pain, benzodiazepine use, and self-reported illicit drug use. Less than 50% of patients reported use of non-opiate adjunct pain medications. No association was observed between PreCOU and pre- or postoperative ABI, or number of prior lower extremity interventions. Following revascularization, the median ABI was 0.88. PreCOU was not associated with significant differences in postoperative complications, length of stay, or mortality. Overall, 71 patients (35%) met the definition for PostCOU, 14 of whom had no history of preoperative chronic opiate use. Ten patients with PreCOU did not demonstrate PostCOU. CONCLUSIONS: Chronic opiate use was common in patients with PAD with a prevalence of approximately 35%, both prior to and following revascularization. Revascularization was associated with a termination of chronic opiate use in less than 15% of patients with PreCOU. Additionally, 10% of patients who did not use opiates chronically before their revascularization did so afterwards. Patients with PAD requiring intervention represent a high-risk group with regards to chronic opiate use. Increased diligence in identifying opioid use among patients with PAD and optimizing the use of non-narcotic adjunct pain medications may result in a lower prevalence of chronic opiate use and its attendant adverse effects.