Genetic and molecular architecture of complex traits.

Human genetics has emerged as one of the most dynamic areas of biology, with a broadening societal impact. In this review, we discuss recent achievements, ongoing efforts, and future challenges in the field. Advances in technology, statistical methods, and the growing scale of research efforts have all provided many insights into the processes that have given rise to the current patterns of genetic variation. Vast maps of genetic associations with human traits and diseases have allowed characterization of their genetic architecture. Finally, studies of molecular and cellular effects of genetic variants have provided insights into biological processes underlying disease. Many outstanding questions remain, but the field is well poised for groundbreaking discoveries as it increases the use of genetic data to understand both the history of our species and its applications to improve human health.

Trans Effects on Gene Expression Can Drive Omnigenic Inheritance.

Early genome-wide association studies (GWASs) led to the surprising discovery that, for typical complex traits, most of the heritability is due to huge numbers of common variants with tiny effect sizes. Previously, we argued that new models are needed to understand these patterns. Here, we provide a formal model in which genetic contributions to complex traits are partitioned into direct effects from core genes and indirect effects from peripheral genes acting in trans. We propose that most heritability is driven by weak trans-eQTL SNPs, whose effects are mediated through peripheral genes to impact the expression of core genes. In particular, if the core genes for a trait tend to be co-regulated, then the effects of peripheral variation can be amplified such that nearly all of the genetic variance is driven by weak trans effects. Thus, our model proposes a framework for understanding key features of the architecture of complex traits.

Predicting Splicing from Primary Sequence with Deep Learning.

The splicing of pre-mRNAs into mature transcripts is remarkable for its precision, but the mechanisms by which the cellular machinery achieves such specificity are incompletely understood. Here, we describe a deep neural network that accurately predicts splice junctions from an arbitrary pre-mRNA transcript sequence, enabling precise prediction of noncoding genetic variants that cause cryptic splicing. Synonymous and intronic mutations with predicted splice-altering consequence validate at a high rate on RNA-seq and are strongly deleterious in the human population. De novo mutations with predicted splice-altering consequence are significantly enriched in patients with autism and intellectual disability compared to healthy controls and validate against RNA-seq in 21 out of 28 of these patients. We estimate that 9%-11% of pathogenic mutations in patients with rare genetic disorders are caused by this previously underappreciated class of disease variation.

Characterizing the Major Structural Variant Alleles of the Human Genome.

In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity.

An Expanded View of Complex Traits: From Polygenic to Omnigenic.

A central goal of genetics is to understand the links between genetic variation and disease. Intuitively, one might expect disease-causing variants to cluster into key pathways that drive disease etiology. But for complex traits, association signals tend to be spread across most of the genome-including near many genes without an obvious connection to disease. We propose that gene regulatory networks are sufficiently interconnected such that all genes expressed in disease-relevant cells are liable to affect the functions of core disease-related genes and that most heritability can be explained by effects on genes outside core pathways. We refer to this hypothesis as an "omnigenic" model.

Profiling lariat intermediates reveals genetic determinants of early and late co-transcriptional splicing.

Long introns with short exons in vertebrate genes are thought to require spliceosome assembly across exons (exon definition), rather than introns, thereby requiring transcription of an exon to splice an upstream intron. Here, we developed CoLa-seq (co-transcriptional lariat sequencing) to investigate the timing and determinants of co-transcriptional splicing genome wide. Unexpectedly, 90% of all introns, including long introns, can splice before transcription of a downstream exon, indicating that exon definition is not obligatory for most human introns. Still, splicing timing varies dramatically across introns, and various genetic elements determine this variation. Strong U2AF2 binding to the polypyrimidine tract predicts early splicing, explaining exon definition-independent splicing. Together, our findings question the essentiality of exon definition and reveal features beyond intron and exon length that are determinative for splicing timing.

RNA editing underlies genetic risk of common inflammatory diseases.

A major challenge in human genetics is to identify the molecular mechanisms of trait-associated and disease-associated variants. To achieve this, quantitative trait locus (QTL) mapping of genetic variants with intermediate molecular phenotypes such as gene expression and splicing have been widely adopted1,2. However, despite successes, the molecular basis for a considerable fraction of trait-associated and disease-associated variants remains unclear3,4. Here we show that ADAR-mediated adenosine-to-inosine RNA editing, a post-transcriptional event vital for suppressing cellular double-stranded RNA (dsRNA)-mediated innate immune interferon responses5-11, is an important potential mechanism underlying genetic variants associated with common inflammatory diseases. We identified and characterized 30,319 cis-RNA editing QTLs (edQTLs) across 49 human tissues. These edQTLs were significantly enriched in genome-wide association study signals for autoimmune and immune-mediated diseases. Colocalization analysis of edQTLs with disease risk loci further pinpointed key, putatively immunogenic dsRNAs formed by expected inverted repeat Alu elements as well as unexpected, highly over-represented cis-natural antisense transcripts. Furthermore, inflammatory disease risk variants, in aggregate, were associated with reduced editing of nearby dsRNAs and induced interferon responses in inflammatory diseases. This unique directional effect agrees with the established mechanism that lack of RNA editing by ADAR1 leads to the specific activation of the dsRNA sensor MDA5 and subsequent interferon responses and inflammation7-9. Our findings implicate cellular dsRNA editing and sensing as a previously underappreciated mechanism of common inflammatory diseases.

Alignment of single-cell RNA-seq samples without overcorrection using kernel density matching.

Single-cell RNA sequencing (scRNA-seq) technology is poised to replace bulk cell RNA sequencing for many biological and medical applications as it allows users to measure gene expression levels in a cell type-specific manner. However, data produced by scRNA-seq often exhibit batch effects that can be specific to a cell type, to a sample, or to an experiment, which prevent integration or comparisons across multiple experiments. Here, we present Dmatch, a method that leverages an external expression atlas of human primary cells and kernel density matching to align multiple scRNA-seq experiments for downstream biological analysis. Dmatch facilitates alignment of scRNA-seq data sets with cell types that may overlap only partially and thus allows integration of multiple distinct scRNA-seq experiments to extract biological insights. In simulation, Dmatch compares favorably to other alignment methods, both in terms of reducing sample-specific clustering and in terms of avoiding overcorrection. When applied to scRNA-seq data collected from clinical samples in a healthy individual and five autoimmune disease patients, Dmatch enabled cell type-specific differential gene expression comparisons across biopsy sites and disease conditions and uncovered a shared population of pro-inflammatory monocytes across biopsy sites in RA patients. We further show that Dmatch increases the number of eQTLs mapped from population scRNA-seq data. Dmatch is fast, scalable, and improves the utility of scRNA-seq for several important applications. Dmatch is freely available online.

Bletilla striata Polysaccharide-Containing Carboxymethyl Cellulose Bilayer Structure Membrane for Prevention of Postoperative Adhesion and Achilles Tendon Repair.

Postoperative tissue adhesion and poor tendon healing are major clinical problems associated with tendon surgery. To avoid postoperative adhesion and promote tendon healing, we developed and synthesized a membrane to wrap the surgical site after tendon suturing. The bilayer-structured porous membrane comprised an outer layer [1,4-butanediol diglycidyl ether cross-linked with carboxymethyl cellulose (CX)] and an inner layer [1,4-butanediol diglycidyl ether cross-linked with Bletilla striata polysaccharides and carboxymethyl cellulose (CXB)]. The morphology, chemical functional groups, and membrane structure were determined. In vitro experiments revealed that the CX/CXB membrane demonstrated good biosafety and biodegradability, promoted tenocyte proliferation and migration, and exhibited low cell attachment and anti-inflammatory effects. Furthermore, in in vivo animal study, the CX/CXB membrane effectively reduced postoperative tendon-peripheral tissue adhesion and improved tendon repair, downregulating inflammatory cytokines in the tendon tissue at the surgical site, which ultimately increased tendon strength by 54% after 4 weeks.

Pulsar emission amplified and resolved by plasma lensing in an eclipsing binary.

Radio pulsars scintillate because their emission travels through the ionized interstellar medium along multiple paths, which interfere with each other. It has long been realized that, independent of their nature, the regions responsible for the scintillation could be used as 'interstellar lenses' to localize pulsar emission regions1,2. Most such lenses, however, resolve emission components only marginally, limiting results to statistical inferences and detections of small positional shifts3-5. As lenses situated close to their source offer better resolution, it should be easier to resolve emission regions of pulsars located in high-density environments such as supernova remnants 6 or binaries in which the pulsar's companion has an ionized outflow. Here we report observations of extreme plasma lensing in the 'black widow' pulsar, B1957+20, near the phase in its 9.2-hour orbit at which its emission is eclipsed by its companion's outflow7-9. During the lensing events, the observed radio flux is enhanced by factors of up to 70-80 at specific frequencies. The strongest events clearly resolve the emission regions: they affect the narrow main pulse and parts of the wider interpulse differently. We show that the events arise naturally from density fluctuations in the outer regions of the outflow, and we infer a resolution of our lenses that is comparable to the pulsar's radius, about 10 kilometres. Furthermore, the distinct frequency structures imparted by the lensing are reminiscent of what is observed for the repeating fast radio burst FRB 121102, providing observational support for the idea that this source is observed through, and thus at times strongly magnified by, plasma lenses 10 .

Monitoring the transition from corneal ulceration to healed scar using a Scheimpflug tomography-based densitometry.

PURPOSE: To compare corneal haze between active ulcer and healed scarring using a Scheimpflug densitometry. MATERIALS AND METHODS: A prospective longitudinal study enrolled 30 patients (30 eyes) with ulcerative keratitis (UK). Each subject's corneal optical density (COD) was measured with a Scheimpflug corneal densitometry, Pentacam® AXL (Oculus GmbH, Wetzlar, Germany), at the active ulcerative and complete scarring stage. The COD data were analyzed through distinct methods (inbuilt, sorted annular partitions, and ulcer-matching densitometric maps). We compared different CODs to select the better index for clinically monitoring the transition from corneal ulceration to healed scar. RESULTS: The CODs of the periphery (P = 0.0024) and outside of the active ulcer (P = 0.0002) significantly decreased after scarring. Partitioning the cornea into different depths and annular zones, the anterior layer, center layer, and the 2-6 mm annular zone had a more remarkable COD decrease after scar formation. The 3rd-sorted COD in the anterior layer revealed the highest area under the receiver-operating characteristic curves (0.709), in which 90% of subjects had COD reduction during the ulcer-to-scar transition. CONCLUSIONS: Aside from subjective judgment based on clinical signs, the Scheimpflug tomography-based densitometry could provide objective and efficient monitoring of the corneal opacity evolution in UK patients. Because the 3rd-sorted annular COD is a better index than the inbuilt or mapping CODs in differentiating active ulcers from healed scars, this COD could be a clinically promising parameter to monitor the progression of UK patients.

Ultraviolet A at levels experienced outdoors suppresses transforming growth factor-beta signaling and collagen production in human scleral fibroblasts.

Previous studies have highlighted the importance of outdoor time in reducing the risk of myopia progression. Although ultraviolet A (UVA) radiation dominates in terms of energy with respect to the UV radiation reaching the Earth's surface, its effects on the exposed anterior sclera have not been well studied. This study was designed to investigate the UVA-induced biological effects at peak sunlight levels in human scleral fibroblasts (HSFs). Using next-generation sequencing (NGS), we analyzed the differentially expressed genes (DEGs) in UVA-treated and normal HSFs. Further, we then identified the functions and key regulators of the DEGs using bioinformatics analysis, and verified the effects of UVA on gene and protein expression in HSFs using real-time PCR, western blotting, and immunofluorescence imaging. The highest level of solar UVA (365 nm) was 3.4 ± 0.18 (mW/cm2). The results from the functional analysis of the DEGs were related to structural changes in the extracellular matrix (ECM) and protein metabolism. Transforming growth factor-ß1 (TGF-ß1) and Smad3 were predicted to be potential upstream regulators, associated with ECM organization. Exposure to a single wavelength of UVA (365 nm, 3 mW/cm2) for 1 h for 5 consecutive days induced the downregulation of the mRNA of ECM genes including COL1A1, COL3A1, COL5A1, VCAN and collagen I protein in HSF. UVA downregulated Smad3 protein and reduced TGF-ß-induced collagen I protein production following UVA exposure in HSF. In conclusion, high UVA exposure reduces TGF-ß signaling and collagen I production by modulating Smad levels in HSF. The effects of overexposure to high-intensity UVA on myopia control require further investigations.

Current Role and Potential of Polymeric Biomaterials in Clinical Obesity Treatment.

The rise of obesity and associated fatal diseases has taken a massive toll worldwide. Despite the existing pharmaceuticals and bariatric surgeries, these approaches manifest limited efficacy or accompany various side effects. Therefore, researchers seek to facilitate the prolonged and specific delivery of therapeutics. Or else, to mimic the essential part of "gastric bypass" by physically blocking excessive absorption via less invasive methods. To achieve these goals, polymeric biomaterials have gained tremendous interest recently. They are known for synthesizing hydrogels, microneedle patches, mucoadhesive coatings, polymer conjugates, and so forth. In this Review, we provide insights into the current studies of polymeric biomaterials in the prevention and treatment of obesity, inspiring future improvements in this regime of study.

Dexmedetomidine for adult cardiac surgery: a systematic review, meta-analysis and trial sequential analysis.

The effects of dexmedetomidine in adults undergoing cardiac surgery are inconsistent. We conducted a systematic review and meta-analysis to analyse the effects of peri-operative dexmedetomidine in adults undergoing cardiac surgery. We searched MEDLINE via Pubmed, EMBASE, Scopus and Cochrane for relevant randomised controlled trials between 1 January 1990 and 1 March 2022. We used the Joanna Briggs Institute methodology checklist to assess study quality and the GRADE approach to certainty of evidence. We assessed the sensitivity of results to false data. We used random-effects meta-analyses to analyse the primary outcomes: durations of intensive care and tracheal intubation. We included 48 trials of 6273 participants. Dexmedetomidine reduced the mean (95%CI) duration of intensive care by 5.0 (2.2-7.7) h, p = 0.001, and tracheal intubation by 1.6 (0.6-2.7) h, p = 0.003. The relative risk (95%CI) for postoperative delirium was 0.58 (0.43-0.78), p = 0.001; 0.76 (0.61-0.95) for atrial fibrillation, p = 0.015; and 0.49 (0.25-0.97) for short-term mortality, p = 0.041. Bradycardia and hypotension were not significantly affected. Trial sequential analysis was consistent with the primary meta-analysis. Adjustments for possible false data reduced the mean (95%CI) reduction in duration of intensive care and tracheal intubation by dexmedetomidine to 3.6 (1.8-5.4) h and 0.8 (0.2-1.4) h, respectively. Binary adjustment for methodological quality at a Joanna Briggs Institute score threshold of 10 did not alter the results significantly. In summary, peri-operative dexmedetomidine reduced the durations of intensive care and tracheal intubation and the incidence of short-term mortality after adult cardiac surgery. The reductions in intensive care stay and tracheal intubation may or may not be considered clinically useful, particularly after adjustment for possible false data.

The Prognostic Biomarkers of Plasma Trimethylamine N-Oxide and Short-Chain Fatty Acids for Recanalization Therapy in Acute Ischemic Stroke.

Bidirectional communication of the microbiota-gut-brain axis is crucial in stroke. Recanalization therapy, namely intravenous thrombolysis (IVT) and endovascular thrombectomy (EVT), are recommended for eligible patients with acute ischemic stroke (AIS). It remains unclear whether gut microbiota metabolites, namely trimethylamine N-oxide (TMAO) and short-chain fatty acids (SCFAs), can predict the prognosis after recanalization therapy. This prospective study recruited patients with AIS receiving IVT, EVT, or both. The National Institutes of Health Stroke Scale (NIHSS) and modified Rankin scale (mRS) scores were used to assess the severity and functional outcomes of AIS, respectively. A functional outcome of mild-to-moderate disability was defined as a mRS score of 0-3 at discharge. Plasma TMAO and SCFA levels were measured through liquid chromatography with triple-quadrupole mass spectrometry. Fifty-six adults undergoing recanalization therapy for AIS were enrolled. Results showed that TMAO levels were not associated with stroke severity and functional outcomes, while isovalerate levels (one of the SCFAs) were negatively correlated with NIHSS scores at admission and discharge. In addition, high isovalerate levels were independently associated with a decreased likelihood of severe disability. The study concluded that an elevated plasma isovalerate level was correlated with mild stroke severity and disability after recanalization therapy for AIS.

Effects of Fecal Occult Blood Immunoassay Screening for Colorectal Cancer-Experience from a Hospital in Central Taiwan.

Background and Objectives: In 2004, the Health Administration of Taiwan began to promote a hospital-based cancer screening quality improvement program, under the principle that "prevention is better than therapy". The aim of this study was to evaluate the effectiveness of colorectal cancer (CRC) screening in patients who received a fecal immunochemical test (FIT) at a hospital in central Taiwan. Materials and Methods: This was a retrospective study. Results: Fecal occult blood immunoassays for CRC screening were conducted in 58,891 participants, of whom 6533 were positive (positive detection rate 11.10%). The positive patients then underwent colonoscopy, and the detection rates of polyps and CRC accounted for 53.6% and 2.4% of all colonoscopy-confirmed diagnoses (3607), respectively. We further enrolled data from patients diagnosed with CRC at our hospital from 2010 to 2018. The patients with CRC were divided into two groups according to whether or not they had received fecal occult blood screening. Among the 88 patients with CRC by screening, 54 had detailed medical records including cancer stage. Of these 54 patients, 1 (1.8%) had pre-stage, 11 (20.4%) had stage I, 24 (44.4%) had stage II, 10 (18.5%) had stage III, and 8 (14.8%) had stage IV CRC. The early cancer detection rates of the screening and non-screening groups were 66.7% and 52.7%, respectively, and the difference was significant (p = 0.00130). Conclusions: In this study, screening with FIT significantly increased the early detection of CRC. The main advantage of FIT is the non-invasiveness and low cost. It is hoped that the further adoption of early screening can increase the detection rates of colorectal polyps or early cancer to improve survival, reduce the high cost of subsequent cancer treatment, and reduce the burden on the patient and healthcare system.

Discovery and characterization of variance QTLs in human induced pluripotent stem cells.

Quantification of gene expression levels at the single cell level has revealed that gene expression can vary substantially even across a population of homogeneous cells. However, it is currently unclear what genomic features control variation in gene expression levels, and whether common genetic variants may impact gene expression variation. Here, we take a genome-wide approach to identify expression variance quantitative trait loci (vQTLs). To this end, we generated single cell RNA-seq (scRNA-seq) data from induced pluripotent stem cells (iPSCs) derived from 53 Yoruba individuals. We collected data for a median of 95 cells per individual and a total of 5,447 single cells, and identified 235 mean expression QTLs (eQTLs) at 10% FDR, of which 79% replicate in bulk RNA-seq data from the same individuals. We further identified 5 vQTLs at 10% FDR, but demonstrate that these can also be explained as effects on mean expression. Our study suggests that dispersion QTLs (dQTLs) which could alter the variance of expression independently of the mean can have larger fold changes, but explain less phenotypic variance than eQTLs. We estimate 4,015 individuals as a lower bound to achieve 80% power to detect the strongest dQTLs in iPSCs. These results will guide the design of future studies on understanding the genetic control of gene expression variance.

Frozen Fat Grafts Maintain Vascular Endothelial Growth Factor Expression and Mediate Angiogenesis During Adipose-Derived Stem Cell Enrichment for Soft Tissue Augmentation.

BACKGROUND: Fresh fat grafts are commonly used in both esthetic and reconstructive surgeries, but the graft resorption rate varies. Cryopreservation of unused fat for later touch-up is one option to resolve this variation. In our previous studies, we found that fat cryopreservation may be a practical strategy for storing fat tissue. To explore the cryopreservation method, we evaluated the role of vascular endothelial growth factor (VEGF) in human frozen fat grafts. METHODS: The concentration of VEGF in human frozen fat grafts subjected to different preservation times was determined using Western blotting and enzyme-linked immunosorbent assay. The angiogenic effect of frozen fat grafts was evaluated using a chorioallantoic membrane assay. Furthermore, the impact of adding human adipose-derived stem cells (hADSCs) or different concentrations of avastin (bevacizumab) to frozen fat grafts on angiogenesis was assessed. The viability of frozen fat grafts with or without hADSCs was evaluated using a nude mouse implantation study. Explanted fat tissues were examined on days 1, 4, 7, 14, 28, and 90, and morphological and histological analyses, immunohistochemistry, and enzyme-linked immunosorbent assay (VEGF concentration) were carried out. RESULTS: No significant difference in VEGF concentration between fresh and frozen fat was observed with respect to preservation duration. In the chorioallantoic membrane assay, frozen fat grafts with hADSCs displayed significantly enhanced angiogenesis. Avastin was found to decrease angiogenesis in frozen fat grafts. However, in the nude mouse implantation study, frozen fat grafts displayed VEGF maintenance, with the highest concentration observed on day 7. Adding hADSCs to the graft further increased the VEGF concentration and CD31 expression. Fat graft viability was found to be higher in the frozen fat grafts containing hADSCs than in grafts without hADSCs. CONCLUSIONS: Human fat grafts can maintain VEGF expression under frozen conditions for at least 12 months. The addition of hADSCs to the frozen fat graft could further enhance angiogenesis, VEGF expression, and fat cell viability.

A Polysaccharide Isolated from the Herb Bletilla striata Combined with Methylcellulose to Form a Hydrogel via Self-Assembly as a Wound Dressing.

The Bletilla striata Polysaccharide (BSP), a natural polysaccharide derived from the east Asian terrestrial orchid Bletilla striata, is an anti-inflammatory, antiviral, and antioxidant polysaccharide. Traditionally, it has been used to treat hemostasis and for wound healing. In this study, BSP was blended with methylcellulose (MC) and methylparaben (MP) to create a hydrogel through a self-assembly route as a wound dressing. The developed hydrogels were designed as M2Bx, M5Bx, and M8Bx. M stands for MC, and the number represents a percentage. Whereas the second letter of B stands for BSP, and x refers to the percentage variation of BSP: x = 0.5%, 1%, and 2%. All the developed MB hydrogels contained ß-glucopyranosyl and α-mannopyranosyl, and rheology test had a tan δ value ≥ 0.5. The pore sizes of the hydrogels decreased by increasing the MC and BSP content, and they had better properties with respect to water loss and their swelling ratio. Evaluations in vitro and in vivo showed that all of the developed MB hydrogels have good cell viability and wound-healing properties. The M8B2 hydrogel group was found to be superior to the others from within the developed MB hydrogels. Therefore, we believe that the M8B2 hydrogel formulation has a high potential for development as a wound dressing.

Impact of regulatory variation across human iPSCs and differentiated cells.

Induced pluripotent stem cells (iPSCs) are an essential tool for studying cellular differentiation and cell types that are otherwise difficult to access. We investigated the use of iPSCs and iPSC-derived cells to study the impact of genetic variation on gene regulation across different cell types and as models for studies of complex disease. To do so, we established a panel of iPSCs from 58 well-studied Yoruba lymphoblastoid cell lines (LCLs); 14 of these lines were further differentiated into cardiomyocytes. We characterized regulatory variation across individuals and cell types by measuring gene expression levels, chromatin accessibility, and DNA methylation. Our analysis focused on a comparison of inter-individual regulatory variation across cell types. While most cell-type-specific regulatory quantitative trait loci (QTLs) lie in chromatin that is open only in the affected cell types, we found that 20% of cell-type-specific regulatory QTLs are in shared open chromatin. This observation motivated us to develop a deep neural network to predict open chromatin regions from DNA sequence alone. Using this approach, we were able to use the sequences of segregating haplotypes to predict the effects of common SNPs on cell-type-specific chromatin accessibility.