Function and constraint in enhancers with multiple evolutionary origins

Motivation: Thousands of human gene regulatory enhancers are composed of sequences with multiple evolutionary origins. These evolutionarily "complex" enhancers consist of older "core" sequences and younger "derived" sequences. However, the functional relationship between the sequences of different evolutionary origins within complex enhancers is poorly understood. Results: We evaluated the function, selective pressures, and sequence variation across core and derived components of human complex enhancers. We find that both components are older than expected from the genomic background, and cores are enriched for derived sequences of similar evolutionary ages. Both components show strong evidence of biochemical activity in massively parallel report assays (MPRAs). However, core and derived sequences have distinct transcription factor (TF) binding preferences that are largely stable across evolutionary origins. Given these signatures of function, both core and derived sequences have substantial evidence of purifying selection. Nonetheless, derived sequences exhibit weaker purifying selection than adjacent cores. Derived sequences also tolerate more common genetic variation and are enriched compared to cores for eQTL associated with gene expression variability in human populations. Conclusions: Both core and derived sequences have strong evidence of gene regulatory function, but derived sequences have distinct constraint profiles, TF binding preferences, and tolerance to variation compared with cores. We propose that the step-wise integration of younger derived and older core sequences has generated regulatory substrates with robust activity and the potential for functional variation. Our analyses demonstrate that synthesizing study of enhancer evolution and function can aid interpretation of regulatory sequence activity and functional variation across human populations.

Evaluating Slow Pyrolysis of Parthenium hysterophorus Biochar: Perspectives to Acidic Soil Amelioration and Growth of Selected Wheat (Triticum aestivum) Varieties

Application of biochar on acidic soils may improve soil fertility and crop productivity. This study aimed to explore the relevance of parthenium biochar-induced changes in the physicochemical properties and agronomic performance of the selected wheat varieties in acidic soils. A pot trial was used in determining the effect of slow pyrolysis parthenium biochar on acidic soils and the agronomic performance of wheat varieties. A general linear model (GLM) of multivariate analysis and principal component analysis (PCA) was used to compare functional variation among soil assayed parameters with biochar dosages and years. Biochar-treated acidic soils did not show significant differences in their physical properties. However, a significant incremental trend was observed in the soil moisture content. The biochar-amended acidic soils showed noticeable differences in the soil pH, available phosphorous, and exchangeable bases (Ca, K, and Na) compared to the control. In all soil samples, a decreasing trend in the soil micronutrients was observed with an increase in the biochar amounts. The analysis also unveiled significant changes in root length, root and shoot dry biomass, and plant height of wheat varieties in response to the biochar amendments. The application of 19.5 t/ha and 23 t/ha dosages of biochar gave the maximum changes in the agronomic performance of Kekeba and Ogolcha varieties, while the minimum was obtained in the 26.5 t/ha and the control. Furthermore, PCA axis 1 accounted for 74.34% of the total variance within a higher eigenvector value (10.4076), and most of the soil parameters were positively correlated with CEC (0.29), available phosphorous (0.29), and soil pH (0.28); however, the micronutrients were negatively correlated. In conclusion, Parthenium hysterophorus biochar has the potential to amend acidic soils, and thus, the application of 16.0, 19.5, and 23 t·ha−1 biochar dosages are considered suitable to reduce the soil acidity level and improve the agronomic performance of wheat varieties. However, extensive research will be needed to determine the effects of biochar on soil properties and crop production in field conditions.

Rare coding variation illuminates the allelic architecture, risk genes, cellular expression patterns, and phenotypic context of autism

Individuals with autism spectrum disorder (ASD) or related neurodevelopmental disorders (NDDs) often carry disruptive mutations in genes that are depleted of functional variation in the broader population. We build upon this observation and exome sequencing from 154,842 individuals to explore the allelic diversity of rare protein-coding variation contributing risk for ASD and related NDDs. Using an integrative statistical model, we jointly analyzed rare protein-truncating variants (PTVs), damaging missense variants, and copy number variants (CNVs) derived from exome sequencing of 63,237 individuals from ASD cohorts. We discovered 71 genes associated with ASD at a false discovery rate (FDR) ≤ 0.001, a threshold approximately equivalent to exome-wide significance, and 183 genes at FDR ≤ 0.05. Associations were predominantly driven by de novo PTVs, damaging missense variants, and CNVs: 57.4%, 21.2%, and 8.32% of evidence, respectively. Though fewer in number, CNVs conferred greater relative risk than PTVs, and repeat-mediated de novo CNVs exhibited strong maternal bias in parent-of-origin (e.g., 92.3% of 16p11.2 CNVs), whereas all other CNVs showed a paternal bias. To explore how genes associated with ASD and NDD overlap or differ, we analyzed our ASD cohort alongside a developmental delay (DD) cohort from the deciphering developmental disorders study (DDD; n=91,605 samples). We first reanalyzed the DDD dataset using the same models as the ASD cohorts, then performed joint analyses of both cohorts and identified 373 genes contributing to NDD risk at FDR ≤ 0.001 and 662 NDD risk genes at FDR ≤ 0.05. Of these NDD risk genes, 54 genes (125 genes at FDR ≤ 0.05) were unique to the joint analyses and not significant in either cohort alone. Our results confirm overlap of most ASD and DD risk genes, although many differ significantly in frequency of mutation. Analyses of single-cell transcriptome datasets showed that genes associated predominantly with DD were strongly enriched for earlier neurodevelopmental cell types, whereas genes displaying stronger evidence for association in ASD cohorts were more enriched for maturing neurons. The ASD risk genes were also enriched for genes associated with schizophrenia from a separate rare coding variant analysis of 121,570 individuals, emphasizing that these neuropsychiatric disorders share common pathways to risk.

Whole genome sequencing reveals large deletions and other loss of function mutations in Mycobacterium tuberculosis drug resistance genes

Drug resistance in Mycobacterium tuberculosis , the causative agent of tuberculosis disease, arises from genetic mutations in genes coding for drug-targets or drug-converting enzymes. SNPs linked to drug resistance have been extensively studied and form the basis of molecular diagnostics and sequencing-based resistance profiling. However, alternative forms of functional variation such as large deletions and other loss of function (LOF) mutations have received much less attention, but if incorporated into diagnostics they are likely to improve their predictive performance. Our work aimed to characterize the contribution of LOF mutations found in 42 established drug resistance genes linked to 19 anti-tuberculous drugs across 32689 sequenced clinical isolates. The analysed LOF mutations included large deletions (n=586), frameshifts (n=4764) and premature stop codons (n=826). We found LOF mutations in genes strongly linked to pyrazinamide (pncA), isoniazid (katG), capreomycin (tlyA), streptomycin (e.g. gid) and ethionamide (ethA, mshA) (P<10−5), but also in some loci linked to drugs where relatively less phenotypic data is available [e.g. cycloserine, delaminid, bedaquiline, para-aminosalicylic acid (PAS), and clofazimine]. This study reports that large deletions (median size 1115 bp) account for a significant portion of resistance variants found for PAS (+7.1% of phenotypic resistance percentage explained), pyrazinamide (+3.5%) and streptomycin (+2.6%) drugs, and can be used to improve the prediction of cryptic resistance. Overall, our work highlights the importance of including LOF mutations (e.g. large deletions) in predicting genotypic drug resistance, thereby informing tuberculosis infection control and clinical decision-making.

788 Reverse McConnell’s sign in biventricular Takotsubo cardiomyopathy: how echo can guide the diagnosis

An 89-years-old woman presented at Emergency Department with a 10-h history of vertigo, headache, nausea, fatigue, and general discomfort. No chest pain or shortness of breath were reported. She had a history of hypertension, chronic kidney disease, paroxysmal atrial fibrillation (AF), osteoporosis, and hypoacusia. The patient suffered of chronic anxiety and the caregiver referred for a recent and acute emotional distress. At the admission, the patient didn’t show clinical signs of peripheral hypoperfusion. Fine crackles at lungs bases were objectivable with coherent ultra-sound lung comets and mild bilateral pleural effusion. Her usual therapy included nebivolol, apixaban, torsemide, candesartan, and D-vitamin. The EKG showed AF with a heart rate of about 110 b.p.m., no ST-segment deviation and normal QTc. The echo findings showed a slight increase in left ventricle volume with a severe reduction of the ejection fraction due to the akinesia of all apical segments with the typical aspect of the ‘apical ballooning’ and concomitant hyperkinesia of the basal segments. Despite normal dimensions, also the right ventricle showed a peculiar contractile pattern, with hyperkinetic basal movement and akinesia of the apex with the hinge point located in the free wall portion in continuity with the LV septal wall. No significant valvular disease was documented except for moderate tricuspid regurgitation. High-sensitive I troponin peaked up to 1500 pg/ml. The clinical appearance was very suggestive of TTS but INTERTAK score of 61 was not diagnostic and, according to the most recent consensus document, a coronary angiography was performed, without documentation of coronary artery disease. During the hospitalization serial electrocardiographic monitoring showed significant and transient QTc prolongation and dynamic T wave changes resulting in progressive INTERTAK score increase. No ventricular arrhythmic events occurred. The patient was treated with careful fluid support and with beta-blockers for AF rate control. Multiple echocardiographic evaluations documented a progressive recovery of systolic function up to complete normalization of biventricular global and regional systolic function. Clinical data, instrumental evidences and dynamic evolution oriented the diagnosis towards TTS with unusual and uneven impairment of right and left ventricular function. The described case focuses the attention on the reverse McConnell’s sign, an echocardiographic finding not often described in the literature, consisting of akinetic right ventricle apical segment and hyperkinetic basal and mid free wall. This discordant motion is exactly opposite to the classic echocardiographic RV aspect detected in acute significative pulmonary embolism described as McConnell’s sign, hence the name. It has been suggested that this functional variation might be a self-protection system of the heart through a mechanism of hibernation that is similar to that occurring during chronic hypoxia, consisting in a decrease in the ATP utilization and O2 consumption, as suggested by the activation of intracellular β2-induced signalling patterns documented in TTS. Recognizing this finding it’s important not only because it has been associated with a higher risk of developing haemodynamic instability but also to orient working diagnosis of TTS when initial clinical assessment through the INTERTAK score is inconclusive.

Selection shapes the landscape of functional variation in wild house mice

Background Through human-aided dispersal over the last ~ 10,000 years, house mice (Mus musculus) have recently colonized diverse habitats across the globe, promoting the emergence of new traits that confer adaptive advantages in distinct environments. Despite their status as the premier mammalian model system, the impact of this demographic and selective history on the global patterning of disease-relevant trait variation in wild mouse populations is poorly understood. Results Here, we leveraged 154 whole-genome sequences from diverse wild house mouse populations to survey the geographic organization of functional variation and systematically identify signals of positive selection. We show that a significant proportion of wild mouse variation is private to single populations, including numerous predicted functional alleles. In addition, we report strong signals of positive selection at many genes associated with both complex and Mendelian diseases in humans. Notably, we detect a significant excess of selection signals at disease-associated genes relative to null expectations, pointing to the important role of adaptation in shaping the landscape of functional variation in wild mouse populations. We also uncover strong signals of selection at multiple genes involved in starch digestion, including Mgam and Amy1. We speculate that the successful emergence of the human-mouse commensalism may have been facilitated, in part, by dietary adaptations at these loci. Finally, our work uncovers multiple cryptic structural variants that manifest as putative signals of positive selection, highlighting an important and under-appreciated source of false-positive signals in genome-wide selection scans. Conclusions Overall, our findings highlight the role of adaptation in shaping wild mouse genetic variation at human disease-associated genes. Our work also highlights the biomedical relevance of wild mouse genetic diversity and underscores the potential for targeted sampling of mice from specific populations as a strategy for developing effective new mouse models of both rare and common human diseases.

Hetero/Homo-Complexes of Sucrose Transporters May Be a Subtle Mode to Regulate Sucrose Transportation in Grape Berries

The sugar distribution mechanism in fruits has been the focus of research worldwide; however, it remains unclear. In order to elucidate the relevant mechanisms in grape berries, the expression, localization, function, and regulation of three sucrose transporters were studied in three representative Vitis varieties. Both SUC11 and SUC12 expression levels were positively correlated with sugar accumulation in grape berries, whereas SUC27 showed a negative relationship. The alignment analysis and sucrose transport ability of isolated SUCs were determined to reflect coding region variations among V. vinifera, V. amurensis Ruper, and V. riparia, indicating that functional variation existed in one SUT from different varieties. Furthermore, potentially oligomerized abilities of VvSUCs colocalized in the sieve elements of the phloem as plasma membrane proteins were verified. The effects of oligomerization on transport properties were characterized in yeast. VvSUC11 and VvSUC12 are high-affinity/low-capacity types of SUTs that stimulate each other by upregulating Vmax and Km, inhibiting sucrose transport, and downregulating the Km of VvSUC27. Thus, changes in the distribution of different SUTs in the same cell govern functional regulation. The activation and inhibition of sucrose transport could be achieved in different stages and tissues of grape development to achieve an effective distribution of sugar.

Genome, Transcriptome, and Germplasm Sequencing Uncovers Functional Variation in the Warm-Season Grain Legume Horsegram Macrotyloma uniflorum (Lam.) Verdc.

Horsegram is a grain legume with excellent nutritional and remedial properties and good climate resilience, able to adapt to harsh environmental conditions. Here, we used a combination of short- and long-read sequencing technologies to generate a genome sequence of 279.12Mb, covering 83.53% of the estimated total size of the horsegram genome, and we annotated 24,521 genes. De novo prediction of DNA repeats showed that approximately 25.04% of the horsegram genome was made up of repetitive sequences, the lowest among the legume genomes sequenced so far. The major transcription factors identified in the horsegram genome were bHLH, ERF, C2H2, WRKY, NAC, MYB, and bZIP, suggesting that horsegram is resistant to drought. Interestingly, the genome is abundant in Bowman–Birk protease inhibitors (BBIs), which can be used as a functional food ingredient. The results of maximum likelihood phylogenetic and estimated synonymous substitution analyses suggested that horsegram is closely related to the common bean and diverged approximately 10.17 million years ago. The double-digested restriction associated DNA (ddRAD) sequencing of 40 germplasms allowed us to identify 3,942 high-quality SNPs in the horsegram genome. A genome-wide association study with powdery mildew identified 10 significant associations similar to the MLO and RPW8.2 genes. The reference genome and other genomic information presented in this study will be of great value to horsegram breeding programs. In addition, keeping the increasing demand for food with nutraceutical values in view, these genomic data provide opportunities to explore the possibility of horsegram for use as a source of food and nutraceuticals.