Proteome-wide association study and functional validation identify novel protein markers for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy, largely due to the paucity of reliable biomarkers for early detection and therapeutic targeting. Existing blood protein biomarkers for PDAC often suffer from replicability issues, arising from inherent limitations such as unmeasured confounding factors in conventional epidemiologic study designs. To circumvent these limitations, we use genetic instruments to identify proteins with genetically predicted levels to be associated with PDAC risk. Leveraging genome and plasma proteome data from the INTERVAL study, we established and validated models to predict protein levels using genetic variants. By examining 8,275 PDAC cases and 6,723 controls, we identified 40 associated proteins, of which 16 are novel. Functionally validating these candidates by focusing on 2 selected novel protein-encoding genes, GOLM1 and B4GALT1, we demonstrated their pivotal roles in driving PDAC cell proliferation, migration, and invasion. Furthermore, we also identified potential drug repurposing opportunities for treating PDAC. SIGNIFICANCE: PDAC is a notoriously difficult-to-treat malignancy, and our limited understanding of causal protein markers hampers progress in developing effective early detection strategies and treatments. Our study identifies novel causal proteins using genetic instruments and subsequently functionally validates selected novel proteins. This dual approach enhances our understanding of PDAC etiology and potentially opens new avenues for therapeutic interventions.

Associations between genetically predicted plasma protein levels and Alzheimer's disease risk: a study using genetic prediction models.

BACKGROUND: Specific peripheral proteins have been implicated to play an important role in the development of Alzheimer's disease (AD). However, the roles of additional novel protein biomarkers in AD etiology remains elusive. The availability of large-scale AD GWAS and plasma proteomic data provide the resources needed for the identification of causally relevant circulating proteins that may serve as risk factors for AD and potential therapeutic targets. METHODS: We established and validated genetic prediction models for protein levels in plasma as instruments to investigate the associations between genetically predicted protein levels and AD risk. We studied 71,880 (proxy) cases and 383,378 (proxy) controls of European descent. RESULTS: We identified 69 proteins with genetically predicted concentrations showing associations with AD risk. The drugs almitrine and ciclopirox targeting ATP1A1 were suggested to have a potential for being repositioned for AD treatment. CONCLUSIONS: Our study provides additional insights into the underlying mechanisms of AD and potential therapeutic strategies.

Identification of blood protein biomarkers associated with prostate cancer risk using genetic prediction models: analysis of over 140,000 subjects.

Prostate cancer (PCa) brings huge public health burden in men. A growing number of conventional observational studies report associations of multiple circulating proteins with PCa risk. However, the existing findings may be subject to incoherent biases of conventional epidemiologic studies. To better characterize their associations, herein, we evaluated associations of genetically predicted concentrations of plasma proteins with PCa risk. We developed comprehensive genetic prediction models for protein levels in plasma. After testing 1308 proteins in 79 194 cases and 61 112 controls of European ancestry included in the consortia of BPC3, CAPS, CRUK, PEGASUS, and PRACTICAL, 24 proteins showed significant associations with PCa risk, including 16 previously reported proteins and eight novel proteins. Of them, 14 proteins showed negative associations and 10 showed positive associations with PCa risk. For 18 of the identified proteins, potential functional somatic changes of encoding genes were detected in PCa patients in The Cancer Genome Atlas (TCGA). Genes encoding these proteins were significantly involved in cancer-related pathways. We further identified drugs targeting the identified proteins, which may serve as candidates for drug repurposing for treating PCa. In conclusion, this study identifies novel protein biomarker candidates for PCa risk, which may provide new perspectives on the etiology of PCa and improve its therapeutic strategies.

Myelodysplastic syndromes disable human CD271+VCAM1+CD146+ niches supporting normal hematopoietic stem/progenitor cells.

Mesenchymal stem/stromal cells (MSCs) within the bone marrow microenvironment (BMME) support normal hematopoietic stem and progenitor cells (HSPCs). However, the heterogeneity of human MSCs has limited the understanding of their contribution to clonal dynamics and evolution to myelodysplastic syndromes (MDS). We combined three MSC cell surface markers, CD271, VCAM-1 (Vascular Cell Adhesion Molecule-1) and CD146, to isolate distinct subsets of human MSCs from bone marrow aspirates of healthy controls (Control BM). Based on transcriptional and functional analysis, CD271+CD106+CD146+ (NGFR+/VCAM1+/MCAM+/Lin-; NVML) cells display stem cell characteristics, are compatible with murine BM-derived Leptin receptor positive MSCs and provide superior support for normal HSPCs. MSC subsets from 17 patients with MDS demonstrated shared transcriptional changes in spite of mutational heterogeneity in the MDS clones, with loss of preferential support of normal HSPCs by MDS-derived NVML cells. Our data provide a new approach to dissect microenvironment-dependent mechanisms regulating clonal dynamics and progression of MDS.

Effectiveness of an 8-Week Physical Activity Intervention Involving Wearable Activity Trackers and an eHealth App: Mixed Methods Study.

BACKGROUND: Health-promotion interventions incorporating wearable technology or eHealth apps can encourage participants to self-monitor and modify their physical activity and sedentary behavior. In 2020, a Calgary (Alberta, Canada) recreational facility developed and implemented a health-promotion intervention (Vivo Play Scientist program) that provided a commercially available wearable activity tracker and a customized eHealth dashboard to participants free of cost. OBJECTIVE: The aim of this study was to independently evaluate the effectiveness of the Vivo Play Scientist program for modifying physical activity and sedentary behavior during the initial 8 weeks of the piloted intervention. METHODS: Our concurrent mixed methods study included a single-arm repeated-measures quasiexperiment and semistructured interviews. Among the 318 eligible participants (≥18 years of age) registered for the program, 87 completed three self-administered online surveys (baseline, T0; 4 weeks, T1; and 8 weeks, T2). The survey captured physical activity, sedentary behavior, use of wearable technology and eHealth apps, and sociodemographic characteristics. Twenty-three participants were recruited using maximal-variation sampling and completed telephone-administered semistructured interviews regarding their program experiences. Self-reported physical activity and sedentary behavior outcomes were statistically compared among the three time points using Friedman tests. Thematic analysis was used to analyze the interview data. RESULTS: The mean age of participants was 39.8 (SD 7.4) years and 75% (65/87) were women. Approximately half of all participants had previously used wearable technology (40/87, 46%) or an eHealth app (43/87, 49%) prior to the intervention. On average, participants reported wearing the activity tracker (Garmin Vivofit4) for 6.4 (SD 1.7) days in the past week at T1 and for 6.0 (SD 2.2) days in the past week at T2. On average, participants reported using the dashboard for 1.6 (SD 2.1) days in the past week at T1 and for 1.0 (SD 1.8) day in the past week at T2. The mean time spent walking at 8 weeks was significantly higher compared with that at baseline (T0 180.34 vs T2 253.79 minutes/week, P=.005), with no significant differences for other physical activity outcomes. Compared to that at baseline, the mean time spent sitting was significantly lower at 4 weeks (T0 334.26 vs T1 260.46 minutes/day, P<.001) and 8 weeks (T0 334.26 vs T2 267.13 minutes/day, P<.001). Significant differences in physical activity and sitting between time points were found among subgroups based on the household composition, history of wearable technology use, and history of eHealth app use. Participants described how wearing the Vivofit4 device was beneficial in helping them to modify physical activity and sedentary behavior. The social support, as a result of multiple members of the same household participating in the program, motivated changes in physical activity. Participants experienced improvements in their mental, physical, and social health. CONCLUSIONS: Providing individuals with free-of-cost commercially available wearable technology and an eHealth app has the potential to support increases in physical activity and reduce sedentary behavior in the short term, even under COVID-19 public health restrictions.

Perceived anxiety and physical activity behaviour changes during the early stages of COVID-19 restrictions in community-dwelling adults in Canada: a cross-sectional study.

OBJECTIVES: The COVID-19 pandemic changed daily routines, including physical activity, which could influence physical and mental health. In our study, we describe physical activity and sedentary behaviour patterns in relation to the pandemic and estimate associations between anxiety and physical activity and sedentary behaviour in community-dwelling adults. DESIGN: Cross-sectional study. SETTING: Calgary, Alberta, Canada. PARTICIPANTS: Between April and June 2020, a random sample of 1124 adults (≥18 years) completed an online questionnaire. PRIMARY AND SECONDARY OUTCOMES: The online questionnaire captured current walking, moderate intensity, vigorous intensity and total physical activity and sedentary behaviour (ie, sitting and leisure-based screen time), perceived relative changes in physical activity, sedentary and social behaviours since the pandemic, perceived seriousness and anxiety related to COVID-19, and sociodemographic characteristics. Differences in sociodemographic characteristics, perceived relative change in behaviour and current physical activity and sedentary behaviour were compared between adults with low and high anxiety. RESULTS: Our sample (n=1047) included more females (60.3%) and fewer older adults (19.2%). Most participants (88.4%) considered COVID-19 as extremely or very serious and one-third (32.9%) felt extremely or very anxious. We found no differences (p>0.05) in current physical activity or sedentary behaviour by anxiety level. The largest perceived change in behaviours included social distancing, driving motor vehicles, use of screen-based devices, watching television and interactions with neighbours. We found anxiety-related differences (p<0.05) in perceived changes in various behaviours. CONCLUSIONS: Changes in physical activity, sedentary behaviour and social behaviour occurred soon after the COVID-19 pandemic was declared, and some of these changes differed among those with low and high anxiety.

A transcriptome-wide association study identifies novel blood-based gene biomarker candidates for Alzheimer's disease risk.

Alzheimer's disease (ad) adversely affects the health, quality of life and independence of patients. There is a critical need to identify novel blood gene biomarkers for ad risk assessment. We performed a transcriptome-wide association study to identify biomarker candidates for ad risk. We leveraged two sets of gene expression prediction models of blood developed using different reference panels and modeling strategies. By applying the prediction models to a meta-GWAS including 71 880 (proxy) cases and 383 378 (proxy) controls, we identified significant associations of genetically determined expression of 108 genes in blood with ad risk. Of these, 15 genes were differentially expressed between ad patients and controls with concordant directions in measured expression data. With evidence from the analyses based on both genetic instruments and directly measured expression levels, this study identifies 15 genes with strong support as biomarkers in blood for ad risk, which may enhance ad risk assessment and mechanism-focused studies.

Impact of the COVID-19 Pandemic on Physical Activity and Sedentary Behaviour: A Qualitative Study in a Canadian City.

Public health measures introduced to combat the COVID-19 pandemic have impacted the physical activity, health, and well-being of millions of people. This grounded theory study explored how the COVID-19 pandemic has affected physical activity and perceptions of health among adults in a Canadian city (Calgary). Twelve adults (50% females; 20-70 years) were interviewed between June and October (2020) via telephone or videoconferencing. Using a maximum variation strategy, participants with a range of sociodemographic characteristics, physical activity levels, and perceptions of seriousness and anxiety related to COVID-19 were selected. Semi-structured interviews captured participant perceptions of how their physical activity and perceptions of health changed during the pandemic. Using thematic analysis, four themes were identified: (1) Disruption to Daily Routines, (2) Changes in Physical Activity, (3) Balancing Health, and (4) Family Life. Participants experienced different degrees of disruption in their daily routines and physical activity based on their individual circumstances (e.g., pre-pandemic physical activity, family life, and access to resources). Although participants faced challenges in modifying their daily routines and physical activity, many adapted. Some participants reported enhanced feelings of well-being. Public health strategies that encourage physical activity and promote health should be supported as they are needed during pandemics, such as COVID-19.

Realtors' Perceptions of Social and Physical Neighborhood Characteristics Associated with Active Living: A Canadian Perspective.

Realtors match home-seekers with neighborhoods that have built and social characteristics they desire to pursue active lifestyles. Studies have yet to explore realtors' perspectives on neighborhood design that supports active living. Using qualitative description, our study was to explore the perceptions and understandings of neighborhood design (walkability, healthy, bike-ability, vibrancy, and livability) among urban residential realtors. Nineteen (6 men; 13 women; average age 48 years) self-identified residential realtors from Calgary, Edmonton, and Lethbridge (Canada) completed semi-structured telephone interviews. Content analysis identified themes from the interview data. Specifically, walkability was described as: perceived preferences, destinations and amenities, and connections; a healthy community was described as: encourages outdoor activities, and promotes social homogeneity; bike-ability was described as: bike-ability attributes, and was controversial; vibrancy was described as: community feel, and evidence of life; and livability was described as: subjective, and preferences and necessities. Our findings can inform the refinement of universal definitions and concepts used to in neighborhood urban design.

Parent anxiety and perceptions of their child's physical activity and sedentary behaviour during the COVID-19 pandemic in Canada.

The public health emergency response to the COVID-19 virus has involved physical distancing strategies to reduce person-to-person transmission. Pandemics, including COVID-19, may influence changes to physical activity and sedentary behaviours among children. However, the role of parent anxiety related to COVID-19 on children's physical activity and sedentary behaviours has yet to be explored. The purpose of this study was to examine the associations between parent COVID-19 anxiety and physical activity and sedentary behaviours among school-aged children (5-17 years) and; to describe these behaviour patterns among school-aged children in relation to the COVID-19 public health emergency response. Between April and June 2020, a random sample of adults (Calgary, Canada) completed an online questionnaire. This sample included 345 parents of at least one school-aged child (80.5% aged 5 to 11 years and 54.9% male). Approximately one-third of parents (35.7%) reported being extremely or very anxious about COVID-19. During this period, most children increased television watching (58.8%), computing or gaming (56.4%), and use of screen-based devices (75.9%). Not surprisingly, given the mandated closure of playgrounds, approximately one-half of children decreased playing at the park (52.7%) and in public spaces (53.7%). Children's physical activity at home either increased (48.8%) or remained unchanged (32.9%). Children of more anxious parents had fewer visits to the park and were more likely to spend ≥2 h/day computing or gaming compared with children of less anxious parents. Strategies to counteract the unintended consequences of the COVID-19 public health measures on parent and child wellbeing are needed.

Mendelian Randomization Analysis of n-6 Polyunsaturated Fatty Acid Levels and Pancreatic Cancer Risk.

BACKGROUND: Whether circulating polyunsaturated fatty acid (PUFA) levels are associated with pancreatic cancer risk is uncertain. Mendelian randomization (MR) represents a study design using genetic instruments to better characterize the relationship between exposure and outcome. METHODS: We utilized data from genome-wide association studies within the Pancreatic Cancer Cohort Consortium and Pancreatic Cancer Case-Control Consortium, involving approximately 9,269 cases and 12,530 controls of European descent, to evaluate associations between pancreatic cancer risk and genetically predicted plasma n-6 PUFA levels. Conventional MR analyses were performed using individual-level and summary-level data. RESULTS: Using genetic instruments, we did not find evidence of associations between genetically predicted plasma n-6 PUFA levels and pancreatic cancer risk [estimates per one SD increase in each PUFA-specific weighted genetic score using summary statistics: linoleic acid odds ratio (OR) = 1.00, 95% confidence interval (CI) = 0.98-1.02; arachidonic acid OR = 1.00, 95% CI = 0.99-1.01; and dihomo-gamma-linolenic acid OR = 0.95, 95% CI = 0.87-1.02]. The OR estimates remained virtually unchanged after adjustment for covariates, using individual-level data or summary statistics, or stratification by age and sex. CONCLUSIONS: Our results suggest that variations of genetically determined plasma n-6 PUFA levels are not associated with pancreatic cancer risk. IMPACT: These results suggest that modifying n-6 PUFA levels through food sources or supplementation may not influence risk of pancreatic cancer.

A Transcriptome-Wide Association Study Identifies Candidate Susceptibility Genes for Pancreatic Cancer Risk.

Pancreatic cancer is among the most well-characterized cancer types, yet a large proportion of the heritability of pancreatic cancer risk remains unclear. Here, we performed a large transcriptome-wide association study to systematically investigate associations between genetically predicted gene expression in normal pancreas tissue and pancreatic cancer risk. Using data from 305 subjects of mostly European descent in the Genotype-Tissue Expression Project, we built comprehensive genetic models to predict normal pancreas tissue gene expression, modifying the UTMOST (unified test for molecular signatures). These prediction models were applied to the genetic data of 8,275 pancreatic cancer cases and 6,723 controls of European ancestry. Thirteen genes showed an association of genetically predicted expression with pancreatic cancer risk at an FDR ≤ 0.05, including seven previously reported genes (INHBA, SMC2, ABO, PDX1, RCCD1, CFDP1, and PGAP3) and six novel genes not yet reported for pancreatic cancer risk [6q27: SFT2D1 OR (95% confidence interval (CI), 1.54 (1.25-1.89); 13q12.13: MTMR6 OR (95% CI), 0.78 (0.70-0.88); 14q24.3: ACOT2 OR (95% CI), 1.35 (1.17-1.56); 17q12: STARD3 OR (95% CI), 6.49 (2.96-14.27); 17q21.1: GSDMB OR (95% CI), 1.94 (1.45-2.58); and 20p13: ADAM33 OR (95% CI): 1.41 (1.20-1.66)]. The associations for 10 of these genes (SFT2D1, MTMR6, ACOT2, STARD3, GSDMB, ADAM33, SMC2, RCCD1, CFDP1, and PGAP3) remained statistically significant even after adjusting for risk SNPs identified in previous genome-wide association study. Collectively, this analysis identified novel candidate susceptibility genes for pancreatic cancer that warrant further investigation. SIGNIFICANCE: A transcriptome-wide association analysis identified seven previously reported and six novel candidate susceptibility genes for pancreatic cancer risk.

Adipose Tissue-Derived Mesenchymal Stem Cells Protect Against Amiodarone-Induced Lung Injury in Rats.

Pulmonary fibrosis (PF) is a progressive and irreversible lung disease, characterized by poor prognosis with limited treatment options. Mesenchymal stem cells (MSCs) are multi-potent cells having the ability to self-renew and differentiate into multiple tissues, thus considered a novel treatment option. The present study aimed to investigate the possible antifibrotic effect of undifferentiated adipose tissue-derived mesenchymal stem cell (AD-MSC) therapy on PF experimentally induced in rats using amiodarone (AMD). AMD (30 mg/kg) was given orally, once daily for 12 consecutive weeks to induce lung fibrosis. Following the confirmation of lung damage with histopathological examination, AD-MSCs (2 × 106 and 4 × 106 undifferentiated MSCs) were injected once intravenously, followed by 2 months for treatment. AMD induced focal fibroblastic cells proliferation in the peribronchiolar tissue, as well as in between the collapsed emphysematous alveoli. Also, AMD significantly increased serum and lung homogenate fibroblast growth factor-7 (FGF7), Clara cell protein-16 (CC16), and cytokeratin -19 (CK19) levels, as well as the expression of both iNOS and NFкB in the lung alveoli. Moreover, AMD caused excessive collagen deposition and increased alpha smooth muscle actin (α-SMA) expression. All findings significantly regressed on stem cell therapy in both doses, with superior effect of the high dose, providing evidence that adipose tissue-derived MSCs could be a promising approach for the treatment of PF. Graphical Abstract.

Conservation of location of several specific inhibitory codon pairs in the Saccharomyces sensu stricto yeasts reveals translational selection.

Synonymous codons provide redundancy in the genetic code that influences translation rates in many organisms, in which overall codon use is driven by selection for optimal codons. It is unresolved if or to what extent translational selection drives use of suboptimal codons or codon pairs. In Saccharomyces cerevisiae, 17 specific inhibitory codon pairs, each comprised of adjacent suboptimal codons, inhibit translation efficiency in a manner distinct from their constituent codons, and many are translated slowly in native genes. We show here that selection operates within Saccharomyces sensu stricto yeasts to conserve nine of these codon pairs at defined positions in genes. Conservation of these inhibitory codon pairs is significantly greater than expected, relative to conservation of their constituent codons, with seven pairs more highly conserved than any other synonymous pair. Conservation is strongly correlated with slow translation of the pairs. Conservation of suboptimal codon pairs extends to two related Candida species, fungi that diverged from Saccharomyces ∼270 million years ago, with an enrichment for codons decoded by I•A and U•G wobble in both Candida and Saccharomyces. Thus, conservation of inhibitory codon pairs strongly implies selection for slow translation at particular gene locations, executed by suboptimal codon pairs.

Identification of new high affinity targets for Roquin based on structural conservation.

Post-transcriptional gene regulation controls the amount of protein produced from a specific mRNA by altering both its decay and translation rates. Such regulation is primarily achieved by the interaction of trans-acting factors with cis-regulatory elements in the untranslated regions (UTRs) of mRNAs. These interactions are guided either by sequence- or structure-based recognition. Similar to sequence conservation, the evolutionary conservation of a UTR's structure thus reflects its functional importance. We used such structural conservation to identify previously unknown cis-regulatory elements. Using the RNA folding program Dynalign, we scanned all UTRs of humans and mice for conserved structures. Characterizing a subset of putative conserved structures revealed a binding site of the RNA-binding protein Roquin. Detailed functional characterization in vivo enabled us to redefine the binding preferences of Roquin and identify new target genes. Many of these new targets are unrelated to the established role of Roquin in inflammation and immune responses and thus highlight additional, unstudied cellular functions of this important repressor. Moreover, the expression of several Roquin targets is highly cell-type-specific. In consequence, these targets are difficult to detect using methods dependent on mRNA abundance, yet easily detectable with our unbiased strategy.

Domestic chickens activate a piRNA defense against avian leukosis virus.

PIWI-interacting RNAs (piRNAs) protect the germ line by targeting transposable elements (TEs) through the base-pair complementarity. We do not know how piRNAs co-evolve with TEs in chickens. Here we reported that all active TEs in the chicken germ line are targeted by piRNAs, and as TEs lose their activity, the corresponding piRNAs erode away. We observed de novo piRNA birth as host responds to a recent retroviral invasion. Avian leukosis virus (ALV) has endogenized prior to chicken domestication, remains infectious, and threatens poultry industry. Domestic fowl produce piRNAs targeting ALV from one ALV provirus that was known to render its host ALV resistant. This proviral locus does not produce piRNAs in undomesticated wild chickens. Our findings uncover rapid piRNA evolution reflecting contemporary TE activity, identify a new piRNA acquisition modality by activating a pre-existing genomic locus, and extend piRNA defense roles to include the period when endogenous retroviruses are still infectious.

De novo mutations in SIK1 cause a spectrum of developmental epilepsies.

Developmental epilepsies are age-dependent seizure disorders for which genetic causes have been increasingly identified. Here we report six unrelated individuals with mutations in salt-inducible kinase 1 (SIK1) in a series of 101 persons with early myoclonic encephalopathy, Ohtahara syndrome, and infantile spasms. Individuals with SIK1 mutations had short survival in cases with neonatal epilepsy onset, and an autism plus developmental syndrome after infantile spasms in others. All six mutations occurred outside the kinase domain of SIK1 and each of the mutants displayed autophosphorylation and kinase activity toward HDAC5. Three mutations generated truncated forms of SIK1 that were resistant to degradation and also showed changes in sub-cellular localization compared to wild-type SIK1. We also report the human neuropathologic examination of SIK1-related developmental epilepsy, with normal neuronal morphology and lamination but abnormal SIK1 protein cellular localization. Therefore, these results expand the genetic etiologies of developmental epilepsies by demonstrating SIK1 mutations as a cause of severe developmental epilepsy.

Novel mutations in ATP1A3 associated with catastrophic early life epilepsy, episodic prolonged apnea, and postnatal microcephaly.

OBJECTIVE: Mutations of ATP1A3 have been associated with rapid onset dystonia-parkinsonism and more recently with alternating hemiplegia of childhood. Here we report one child with catastrophic early life epilepsy and shortened survival, and another with epilepsy, episodic prolonged apnea, postnatal microcephaly, and severe developmental disability. Novel heterozygous mutations (p.Gly358Val and p.Ile363Asn) were identified in ATP1A3 in these children. METHODS: Subjects underwent next-generation sequencing under a research protocol. Clinical data were collected retrospectively. The biochemical effects of the mutations on ATP1A3 protein function were investigated. Postmortem neuropathologic specimens from control and affected subjects were studied. RESULTS: The mutations localized to the P domain of the Na,K-ATPase α3 protein, and resulted in significant reduction of Na,K-ATPase activity in vitro. We demonstrate in both control human brain tissue and that from the subject with the p.Gly358Val mutation that ATP1A3 immunofluorescence is prominently associated with interneurons in the cortex, which may provide some insight into the pathogenesis of the disease. SIGNIFICANCE: The findings indicate these mutations cause severe phenotypes of ATP1A3-related disorder spectrum that include catastrophic early life epilepsy, episodic apnea, and postnatal microcephaly.

Comparison of insertion/deletion calling algorithms on human next-generation sequencing data.

BACKGROUND: Insertions/deletions (indels) are the second most common type of genomic variant and the most common type of structural variant. Identification of indels in next generation sequencing data is a challenge, and algorithms commonly used for indel detection have not been compared on a research cohort of human subject genomic data. Guidelines for the optimal detection of biologically significant indels are limited. We analyzed three sets of human next generation sequencing data (48 samples of a 200 gene target exon sequencing, 45 samples of whole exome sequencing, and 2 samples of whole genome sequencing) using three algorithms for indel detection (Pindel, Genome Analysis Tool Kit's UnifiedGenotyper and HaplotypeCaller). RESULTS: We observed variation in indel calls across the three algorithms. The intersection of the three tools comprised only 5.70% of targeted exon, 19.52% of whole exome, and 14.25% of whole genome indel calls. The majority of the discordant indels were of lower read depth and likely to be false positives. When software parameters were kept consistent across the three targets, HaplotypeCaller produced the most reliable results. Pindel results did not validate well without adjustments to parameters to account for varied read depth and number of samples per run. Adjustments to Pindel's M (minimum support for event) parameter improved both concordance and validation rates. Pindel was able to identify large deletions that surpassed the length capabilities of the GATK algorithms. CONCLUSIONS: Despite the observed variability in indel identification, we discerned strengths among the individual algorithms on specific data sets. This allowed us to suggest best practices for indel calling. Pindel's low validation rate of indel calls made in targeted exon sequencing suggests that HaplotypeCaller is better suited for short indels and multi-sample runs in targets with very high read depth. Pindel allows for optimization of minimum support for events and is best used for detection of larger indels at lower read depths.

De novo mutations in the beta-tubulin gene TUBB2A cause simplified gyral patterning and infantile-onset epilepsy.

Tubulins, and microtubule polymers into which they incorporate, play critical mechanical roles in neuronal function during cell proliferation, neuronal migration, and postmigrational development: the three major overlapping events of mammalian cerebral cortex development. A number of neuronally expressed tubulin genes are associated with a spectrum of disorders affecting cerebral cortex formation. Such "tubulinopathies" include lissencephaly/pachygyria, polymicrogyria-like malformations, and simplified gyral patterns, in addition to characteristic extracortical features, such as corpus callosal, basal ganglia, and cerebellar abnormalities. Epilepsy is a common finding in these related disorders. Here we describe two unrelated individuals with infantile-onset epilepsy and abnormalities of brain morphology, harboring de novo variants that affect adjacent amino acids in a beta-tubulin gene TUBB2A. Located in a highly conserved loop, we demonstrate impaired tubulin and microtubule function resulting from each variant in vitro and by using in silico predictive modeling. We propose that the affected functional loop directly associates with the alpha-tubulin-bound guanosine triphosphate (GTP) molecule, impairing the intradimer interface and correct formation of the alpha/beta-tubulin heterodimer. This study associates mutations in TUBB2A with the spectrum of "tubulinopathy" phenotypes. As a consequence, genetic variations affecting all beta-tubulin genes expressed at high levels in the brain (TUBB2B, TUBB3, TUBB, TUBB4A, and TUBB2A) have been linked with malformations of cortical development.