Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease
. However, organoids lack the connectivity that exists in vivo, which limits ...maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered.
Rhinovirus (RV) is the most prevalent human respiratory virus and is responsible for at least half of all common colds. RV infections may result in a broad spectrum of effects that range from ...asymptomatic infections to severe lower respiratory illnesses. The basis for inter-individual variation in the response to RV infection is not well understood. In this study, we explored whether host genetic variation is associated with variation in gene expression response to RV infections between individuals. To do so, we obtained genome-wide genotype and gene expression data in uninfected and RV-infected peripheral blood mononuclear cells (PBMCs) from 98 individuals. We mapped local and distant genetic variation that is associated with inter-individual differences in gene expression levels (eQTLs) in both uninfected and RV-infected cells. We focused specifically on response eQTLs (reQTLs), namely, genetic associations with inter-individual variation in gene expression response to RV infection. We identified local reQTLs for 38 genes, including genes with known functions in viral response (UBA7, OAS1, IRF5) and genes that have been associated with immune and RV-related diseases (e.g., ITGA2, MSR1, GSTM3). The putative regulatory regions of genes with reQTLs were enriched for binding sites of virus-activated STAT2, highlighting the role of condition-specific transcription factors in genotype-by-environment interactions. Overall, we suggest that the 38 loci associated with inter-individual variation in gene expression response to RV-infection represent promising candidates for affecting immune and RV-related respiratory diseases.
Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types ...that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.
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•Single-cell RNA-seq during cardiac hPSC differentiation reveals cellular heterogeneity•A key cardiac regulatory gene, HOPX, is rarely expressed during in vitro differentiation•HOPX is a key in vitro regulator of cardiomyocyte hypertrophy and maturation
Friedman et al. performed single-cell transcriptional analysis over a time course of in vitro cardiac differentiation from human pluripotent stem cells. They utilized these data to identify the requirement of hypertrophic stimuli for expression of a cardiac regulatory gene, HOPX, to generate cardiomyocytes more accurately reflecting in vivo heart development.
Individualization of cancer management requires prognostic markers and therapy-predictive markers. Prognostic markers assess risk of disease progression independent of therapy, whereas ...therapy-predictive markers identify patients whose disease is sensitive or resistant to treatment. We show that an experimentally derived IFN-related DNA damage resistance signature (IRDS) is associated with resistance to chemotherapy and/or radiation across different cancer cell lines. The IRDS genes STAT1, ISG15, and IFIT1 all mediate experimental resistance. Clinical analyses reveal that IRDS(+) and IRDS(-) states exist among common human cancers. In breast cancer, a seven-gene-pair classifier predicts for efficacy of adjuvant chemotherapy and for local-regional control after radiation. By providing information on treatment sensitivity or resistance, the IRDS improves outcome prediction when combined with standard markers, risk groups, or other genomic classifiers.
Biofouling on the surface of implanted medical devices and biosensors severely hinders device functionality and drastically shortens device lifetime. Poly(ethylene glycol) and zwitterionic polymers ...are currently considered “gold‐standard” device coatings to reduce biofouling. To discover novel anti‐biofouling materials, a combinatorial library of polyacrylamide‐based copolymer hydrogels is created, and their ability is screened to prevent fouling from serum and platelet‐rich plasma in a high‐throughput parallel assay. It is found that certain nonintuitive copolymer compositions exhibit superior anti‐biofouling properties over current gold‐standard materials, and machine learning is used to identify key molecular features underpinning their performance. For validation, the surfaces of electrochemical biosensors are coated with hydrogels and their anti‐biofouling performance in vitro and in vivo in rodent models is evaluated. The copolymer hydrogels preserve device function and enable continuous measurements of a small‐molecule drug in vivo better than gold‐standard coatings. The novel methodology described enables the discovery of anti‐biofouling materials that can extend the lifetime of real‐time in vivo sensing devices.
Hydrogel formulations that prevent protein and platelet adhesion in assays where “gold‐standard” polymers exhibit significant platelet adhesion are identified. By using machine‐learning techniques to analyze molecular features of the polyacrylamide‐based copolymers, features giving rise to this anti‐biofouling performance are identified. Coating electrochemical devices with these top‐performing polyacrylamide‐based hydrogels extend lifetime when implanted intravenously and enable in vivo real‐time monitoring of analytes.
Objective
The objective of this study was to describe molecular findings and phenotypic features among individuals referred for prenatal Beckwith‐Wiedemann syndrome (BWS) testing.
Methods
Molecular ...diagnostic testing was performed using a sensitive quantitative real‐time PCR‐based assay capable of detecting mosaic methylation to the level of 3% at IC1 and IC2. Sanger sequencing of CDKN1C was performed in cases with normal methylation.
Results
Of the 94 patients tested, a molecular diagnosis was identified for 25.5% of cases; 70.9% of diagnosed cases had loss of methylation at IC2, 4.2% had gain of methylation at IC1, 12.5% had paternal uniparental isodisomy, and 12.5% had CDKN1C loss‐of‐function variants. Methylation level changes in prenatal cases were significantly greater than changes identified in cases tested after birth. Cases with a prenatal molecular diagnosis had a significantly greater number of BWS‐associated phenotypic features. The presence of either macroglossia or placentomegaly was most predictive of a BWS diagnosis.
Conclusion
Our results support the consensus statement advocating BWS molecular testing for all patients with one or more BWS‐associated prenatal features and suggest that low‐level mosaic methylation changes may be uncommon among prenatal BWS diagnoses.
Key Points
What is already known about this topic?
Prenatal features and molecular findings associated with prenatal diagnosis of BWS have been previously described in multiple publications.
What does this study add?
This study demonstrates the use of an assay with improved mosaic detection for BWS in a prenatal cohort of patients.
We report which prenatal features are predictive of a diagnosis in our cohort.
Beckwith-Wiedemann Syndrome (BWS) is characterised by overgrowth and tumour predisposition. While multiple epigenetic and genetic mechanisms cause BWS, the majority are caused by methylation defects ...in imprinting control regions on chromosome 11p15.5. Disease-causing methylation defects are often mosaic within affected individuals. Phenotypic variability among individuals with chromosome 11p15.5 defects and tissue mosaicism led to the definition of the Beckwith-Wiedemann Spectrum (BWSp). Molecular diagnosis of BWSp requires use of multiple sensitive diagnostic techniques to reliably detect low-level aberrations.
Multimodal BWS diagnostic testing was performed on samples from 1057 individuals. Testing included use of a sensitive qRT-PCR-based quantitation method enabling identification of low-level mosaic disease, identification of CNVs within 11p15.5 via array comparative genomic hybridisation or qRT-PCR, and Sanger sequencing of
.
A molecular diagnosis was confirmed for 27.4% of individuals tested, of whom 43.4% had mosaic disease. The presence of a single cardinal feature was associated with a molecular diagnosis of BWSp in 20% of cases. Additionally, significant differences in the prevalence of mosaic disease among BWS molecular subtypes were identified. Finally, the diagnostic yield obtained by testing solid tissue samples from individuals with negative blood testing results shows improved molecular diagnosis.
This study highlights the prevalence of mosaic disease among individuals with BWSp and the increases in diagnostic yield obtained via testing both blood and solid tissue samples from affected individuals. Additionally, the results establish the presence of a molecular diagnosis in individuals with very subtle features of BWSp.
Automated Clinical Exome Reanalysis Reveals Novel Diagnoses Baker, Samuel W.; Murrell, Jill R.; Nesbitt, Addie I. ...
The Journal of molecular diagnostics : JMD,
January 2019, 2019-01-00, 20190101, Letnik:
21, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Clinical exome sequencing (CES) has a reported diagnostic yield of 20% to 30% for most clinical indications. The ongoing discovery of novel gene–disease and variant–disease associations are expected ...to increase the diagnostic yield of CES. Performing systematic reanalysis of previously nondiagnostic CES samples represents a significant challenge for clinical laboratories. Here, we present the results of a novel automated reanalysis methodology applied to 300 CES samples initially analyzed between June 2014 and September 2016. Application of our reanalysis methodology reduced reanalysis variant analysis burden by >93% and correctly captured 70 of 70 previously identified diagnostic variants among 60 samples with previously identified diagnoses. Notably, reanalysis of 240 initially nondiagnostic samples using information available on July 1, 2017, revealed 38 novel diagnoses, representing a 15.8% increase in diagnostic yield. Modeling monthly iterative reanalysis of 240 nondiagnostic samples revealed a diagnostic rate of 0.57% of samples per month. Modeling the workload required for monthly iterative reanalysis of nondiagnostic samples revealed a variant analysis burden of approximately 5 variants/month for proband-only and approximately 0.5 variants/month for trio samples. Approximately 45% of samples required evaluation during each monthly interval, and 61.3% of samples were reevaluated across three consecutive reanalyses. In sum, automated reanalysis methods can facilitate efficient reevaluation of nondiagnostic samples using up-to-date literature and can provide significant value to clinical laboratories.
This study aimed to establish variants in CBX1, encoding heterochromatin protein 1β (HP1β), as a cause of a novel syndromic neurodevelopmental disorder.
Patients with CBX1 variants were identified, ...and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays and neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines.
In 3 unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1β, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1β binding to heterochromatin, whereas HP1β interactome analysis demonstrated that the majority of HP1β-interacting proteins remained unchanged between the wild-type and mutant HP1β.
These collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1β chromatin binding during neurocognitive development. Because HP1β forms homodimers and heterodimers, mutant HP1β likely sequesters wild-type HP1β and other HP1 proteins, exerting dominant-negative effects.
Deciphering the impact of genetic variation on gene regulation is fundamental to understanding common, complex human diseases. Although histone modifications are important markers of gene regulatory ...elements of the genome, any specific histone modification has not been assayed in more than a few individuals in the human liver. As a result, the effects of genetic variation on histone modification states in the liver are poorly understood. Here, we generate the most comprehensive genome-wide dataset of two epigenetic marks, H3K4me3 and H3K27ac, and annotate thousands of putative regulatory elements in the human liver. We integrate these findings with genome-wide gene expression data collected from the same human liver tissues and high-resolution promoter-focused chromatin interaction maps collected from human liver-derived HepG2 cells. We demonstrate widespread functional consequences of natural genetic variation on putative regulatory element activity and gene expression levels. Leveraging these extensive datasets, we fine-map a total of 74 GWAS loci that have been associated with at least one complex phenotype. Our results reveal a repertoire of genes and regulatory mechanisms governing complex disease development and further the basic understanding of genetic and epigenetic regulation of gene expression in the human liver tissue.
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