Health-related quality of life and medication use among individuals with Angelman syndrome.

PURPOSE: The primary goal of this analysis is to describe the health-related quality of life (HRQoL), medical history, and medication use among adolescents and adults individuals with Angelman syndrome (AS). METHODS: The analysis uses baseline data collected during the STARS study, a double-blind placebo controlled trial of gaboxadol (OV101) in adolescents and adults with AS. The HRQoL was estimated using EuroQoL 5-Dimension 5-Level (EQ-5D) health questionnaire proxy 1 version, which was completed by the caregivers. EQ-5D consists of two parts, a 5-dimension descriptive and a visual analogue scale (VAS) component. The utility score derived from EQ-5D ranges from 0 to 1 (perfect health) and VAS ranges from 0 to 100 (perfect health). RESULTS: 87 individuals with AS were included in the present analysis. The mean utility score was 0.44 ± 0.20 and VAS score was 84 ± 1.5. The EQ-5D data indicated that the self-care, mobility and daily activities were most impacted. All adolescents (100%) and most adults (93%) had at least moderate problems with self-care activities, such as washing or dressing themselves. More than half (55%) of the adolescents and adults had at least moderate issues with mobility and usual activities. Approximately, 30% of adolescents and adults had moderate to extreme problems with anxiety/depression. High baseline concomitant use of medications was observed across both age groups with an average of 5 medications being used per person. CONCLUSION: This study highlights the impact of AS on HRQoL and medication utilization among adolescents and adults individuals with AS.

Optimizing photoswitchable MEK.

Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both Drosophila and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.

Left-right asymmetric heart jogging increases the robustness of dextral heart looping in zebrafish.

Building a left-right (L-R) asymmetric organ requires asymmetric information. This comes from various sources, including asymmetries in embryo-scale genetic cascades (including the left-sided Nodal cascade), organ-intrinsic mechanical forces, and cell-level chirality, but the relative influence of these sources and how they collaborate to drive asymmetric morphogenesis is not understood. During zebrafish heart development, the linear heart tube extends to the left of the midline in a process known as jogging. The jogged heart then undergoes dextral (i.e. rightward) looping to correctly position the heart chambers relative to one another. Left lateralized jogging is governed by the left-sided expression of Nodal in mesoderm tissue, while looping laterality is mainly controlled by heart-intrinsic cell-level asymmetries in the actomyosin cytoskeleton. The purpose of lateralized jogging is not known. Moreover, after jogging, the heart tube returns to an almost midline position and so it is not clear whether or how jogging may impact the dextral loop. Here, we characterize a novel loss-of-function mutant in the zebrafish Nodal homolog southpaw (spaw) that appears to be a true null. We then assess the relationship between jogging and looping laterality in embryos lacking asymmetric Spaw signals. We found that the probability of a dextral loop occurring, does not depend on asymmetric Spaw signals per se, but does depend on the laterality of jogging. Thus, we conclude that the role of leftward jogging is to spatially position the heart tube in a manner that promotes robust dextral looping. When jogging laterality is abnormal, the robustness of dextral looping decreases. This establishes a cooperation between embryo-scale Nodal-dependent L-R asymmetries and organ-intrinsic cellular chirality in the control of asymmetric heart morphogenesis and shows that the transient laterality of the early heart tube has consequences for later heart morphogenetic events.

ZNRF3 functions in mammalian sex determination by inhibiting canonical WNT signaling.

Mammalian sex determination is controlled by the antagonistic interactions of two genetic pathways: The SRY-SOX9-FGF9 network promotes testis determination partly by opposing proovarian pathways, while RSPO1/WNT-ß-catenin/FOXL2 signals control ovary development by inhibiting SRY-SOX9-FGF9. The molecular basis of this mutual antagonism is unclear. Here we show that ZNRF3, a WNT signaling antagonist and direct target of RSPO1-mediated inhibition, is required for sex determination in mice. XY mice lacking ZNRF3 exhibit complete or partial gonadal sex reversal, or related defects. These abnormalities are associated with ectopic WNT/ß-catenin activity and reduced Sox9 expression during fetal sex determination. Using exome sequencing of individuals with 46,XY disorders of sex development, we identified three human ZNRF3 variants in very rare cases of XY female presentation. We tested two missense variants and show that these disrupt ZNRF3 activity in both human cell lines and zebrafish embryo assays. Our data identify a testis-determining function for ZNRF3 and indicate a mechanism of direct molecular interaction between two mutually antagonistic organogenetic pathways.

Measuring What Matters to Individuals with Angelman Syndrome and Their Families: Development of a Patient-Centered Disease Concept Model.

Angelman syndrome (AS) is a complex, heterogeneous, and life-long neurodevelopmental disorder. Despite the considerable impact on individuals and caregivers, no disease-modifying treatments are available. To support holistic clinical management and the development of AS-specific outcome measures for clinical studies, we conducted primary and secondary research identifying the impact of symptoms on individuals with AS and their unmet need. This qualitative research adopted a rigorous step-wise approach, aggregating information from published literature, then evaluating it via disease concept elicitation interviews with clinical experts and caregivers. We found that the AS-defining concepts most relevant for treatment included: impaired expressive communication, seizures, maladaptive behavior, cognitive impairment, motor function difficulties, sleep disturbance, and limited self-care abilities. We highlight the relevance of age in experiencing these key AS concepts, and the difference between the perceptions of clinicians and caregivers towards the syndrome. Finally, we outline the impact of AS on individuals, caregivers, and families.

Left-Right Patterning: Breaking Symmetry to Asymmetric Morphogenesis.

Vertebrates exhibit striking left-right (L-R) asymmetries in the structure and position of the internal organs. Symmetry is broken by motile cilia-generated asymmetric fluid flow, resulting in a signaling cascade - the Nodal-Pitx2 pathway - being robustly established within mesodermal tissue on the left side only. This pathway impinges upon various organ primordia to instruct their side-specific development. Recently, progress has been made in understanding both the breaking of embryonic L-R symmetry and how the Nodal-Pitx2 pathway controls lateralized cell differentiation, migration, and other aspects of cell behavior, as well as tissue-level mechanisms, that drive asymmetries in organ formation. Proper execution of asymmetric organogenesis is critical to health, making furthering our understanding of L-R development an important concern.

In vivo severity ranking of Ras pathway mutations associated with developmental disorders.

Germ-line mutations in components of the Ras/MAPK pathway result in developmental disorders called RASopathies, affecting about 1/1,000 human births. Rapid advances in genome sequencing make it possible to identify multiple disease-related mutations, but there is currently no systematic framework for translating this information into patient-specific predictions of disease progression. As a first step toward addressing this issue, we developed a quantitative, inexpensive, and rapid framework that relies on the early zebrafish embryo to assess mutational effects on a common scale. Using this assay, we assessed 16 mutations reported in MEK1, a MAPK kinase, and provide a robust ranking of these mutations. We find that mutations found in cancer are more severe than those found in both RASopathies and cancer, which, in turn, are generally more severe than those found only in RASopathies. Moreover, this rank is conserved in other zebrafish embryonic assays and Drosophila-specific embryonic and adult assays, suggesting that our ranking reflects the intrinsic property of the mutant molecule. Furthermore, this rank is predictive of the drug dose needed to correct the defects. This assay can be readily used to test the strengths of existing and newly found mutations in MEK1 and other pathway components, providing the first step in the development of rational guidelines for patient-specific diagnostics and treatment of RASopathies.

How activating mutations affect MEK1 regulation and function.

The MEK1 kinase directly phosphorylates ERK2, after the activation loop of MEK1 is itself phosphorylated by Raf. Studies over the past decade have revealed a large number of disease-related mutations in the MEK1 gene that lead to tumorigenesis and abnormal development. Several of these mutations result in MEK1 constitutive activity, but how they affect MEK1 regulation and function remains largely unknown. Here, we address these questions focusing on two pathogenic variants of the Phe-53 residue, which maps to the well-characterized negative regulatory region of MEK1. We found that these variants are phosphorylated by Raf faster than the wild-type enzyme, and this phosphorylation further increases their enzymatic activity. However, the maximal activities of fully phosphorylated wild-type and mutant enzymes are indistinguishable. On the basis of available structural information, we propose that the activating substitutions destabilize the inactive conformation of MEK1, resulting in its constitutive activity and making it more prone to Raf-mediated phosphorylation. Experiments in zebrafish revealed that the effects of activating variants on embryonic development reflect the joint control of the negative regulatory region and activating phosphorylation. Our results underscore the complexity of the effects of activating mutations on signaling systems, even at the level of a single protein.

CCDC151 mutations cause primary ciliary dyskinesia by disruption of the outer dynein arm docking complex formation.

A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151(ts272a) and mouse Ccdc151(Snbl) mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.

Left-right asymmetry: lessons from Cancún.

The satellite symposium on 'Making and breaking the left-right axis: implications of laterality in development and disease' was held in June 2013 in conjunction with the 17th International Society for Developmental Biology meeting in Cancún, Mexico. As we summarize here, leaders in the field gathered at the symposium to discuss recent advances in understanding how left-right asymmetry is generated and utilized across the animal kingdom.

Integration of nodal and BMP signals in the heart requires FoxH1 to create left-right differences in cell migration rates that direct cardiac asymmetry.

Failure to properly establish the left-right (L/R) axis is a major cause of congenital heart defects in humans, but how L/R patterning of the embryo leads to asymmetric cardiac morphogenesis is still unclear. We find that asymmetric Nodal signaling on the left and Bmp signaling act in parallel to establish zebrafish cardiac laterality by modulating cell migration velocities across the L/R axis. Moreover, we demonstrate that Nodal plays the crucial role in generating asymmetry in the heart and that Bmp signaling via Bmp4 is dispensable in the presence of asymmetric Nodal signaling. In addition, we identify a previously unappreciated role for the Nodal-transcription factor FoxH1 in mediating cell responsiveness to Bmp, further linking the control of these two pathways in the heart. The interplay between these TGFß pathways is complex, with Nodal signaling potentially acting to limit the response to Bmp pathway activation and the dosage of Bmp signals being critical to limit migration rates. These findings have implications for understanding the complex genetic interactions that lead to congenital heart disease in humans.

Two additional midline barriers function with midline lefty1 expression to maintain asymmetric Nodal signaling during left-right axis specification in zebrafish.

Left-right (L/R) patterning is crucial for the proper development of all vertebrates and requires asymmetric expression of nodal in the lateral plate mesoderm (LPM). The mechanisms governing asymmetric initiation of nodal have been studied extensively, but because Nodal is a potent activator of its own transcription, it is also crucial to understand the regulation required to maintain this asymmetry once it is established. The 'midline barrier', consisting of lefty1 expression, is a conserved mechanism for restricting Nodal activity to the left. However, the anterior and posterior extremes of the LPM are competent to respond to Nodal signals yet are not adjacent to this barrier, suggesting that lefty1 is not the only mechanism preventing ectopic Nodal activation. Here, we demonstrate the existence of two additional midline barriers. The first is a 'posterior barrier' mediated by Bmp signaling that prevents nodal propagation through the posterior LPM. In contrast to previous reports, we find that Bmp represses Nodal signaling independently of lefty1 expression and through the activity of a ligand other than Bmp4. The 'anterior barrier' is mediated by lefty2 expression in the left cardiac field and prevents Nodal activation from traveling across the anterior limit of the notochord and propagating down the right LPM. Both barriers appear to be conserved across model systems and are thus likely to be present in all vertebrates.

Functional knowledge transfer for high-accuracy prediction of under-studied biological processes.

A key challenge in genetics is identifying the functional roles of genes in pathways. Numerous functional genomics techniques (e.g. machine learning) that predict protein function have been developed to address this question. These methods generally build from existing annotations of genes to pathways and thus are often unable to identify additional genes participating in processes that are not already well studied. Many of these processes are well studied in some organism, but not necessarily in an investigator's organism of interest. Sequence-based search methods (e.g. BLAST) have been used to transfer such annotation information between organisms. We demonstrate that functional genomics can complement traditional sequence similarity to improve the transfer of gene annotations between organisms. Our method transfers annotations only when functionally appropriate as determined by genomic data and can be used with any prediction algorithm to combine transferred gene function knowledge with organism-specific high-throughput data to enable accurate function prediction. We show that diverse state-of-art machine learning algorithms leveraging functional knowledge transfer (FKT) dramatically improve their accuracy in predicting gene-pathway membership, particularly for processes with little experimental knowledge in an organism. We also show that our method compares favorably to annotation transfer by sequence similarity. Next, we deploy FKT with state-of-the-art SVM classifier to predict novel genes to 11,000 biological processes across six diverse organisms and expand the coverage of accurate function predictions to processes that are often ignored because of a dearth of annotated genes in an organism. Finally, we perform in vivo experimental investigation in Danio rerio and confirm the regulatory role of our top predicted novel gene, wnt5b, in leftward cell migration during heart development. FKT is immediately applicable to many bioinformatics techniques and will help biologists systematically integrate prior knowledge from diverse systems to direct targeted experiments in their organism of study.

Nodal-dependent mesendoderm specification requires the combinatorial activities of FoxH1 and Eomesodermin.

Vertebrate mesendoderm specification requires the Nodal signaling pathway and its transcriptional effector FoxH1. However, loss of FoxH1 in several species does not reliably cause the full range of loss-of-Nodal phenotypes, indicating that Nodal signals through additional transcription factors during early development. We investigated the FoxH1-dependent and -independent roles of Nodal signaling during mesendoderm patterning using a novel recessive zebrafish FoxH1 mutation called midway, which produces a C-terminally truncated FoxH1 protein lacking the Smad-interaction domain but retaining DNA-binding capability. Using a combination of gel shift assays, Nodal overexpression experiments, and genetic epistasis analyses, we demonstrate that midway more accurately represents a complete loss of FoxH1-dependent Nodal signaling than the existing zebrafish FoxH1 mutant schmalspur. Maternal-zygotic midway mutants lack notochords, in agreement with FoxH1 loss in other organisms, but retain near wild-type expression of markers of endoderm and various nonaxial mesoderm fates, including paraxial and intermediate mesoderm and blood precursors. We found that the activity of the T-box transcription factor Eomesodermin accounts for specification of these tissues in midway embryos. Inhibition of Eomesodermin in midway mutants severely reduces the specification of these tissues and effectively phenocopies the defects seen upon complete loss of Nodal signaling. Our results indicate that the specific combinations of transcription factors available for signal transduction play critical and separable roles in determining Nodal pathway output during mesendoderm patterning. Our findings also offer novel insights into the co-evolution of the Nodal signaling pathway, the notochord specification program, and the chordate branch of the deuterostome family of animals.

The exocyst protein Sec10 interacts with Polycystin-2 and knockdown causes PKD-phenotypes.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown-including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation-suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes.

Cooperation between Nodal and FGF signals regulates zebrafish cardiac cell migration and heart morphogenesis.

Asymmetric vertebrate heart development is driven by an intricate sequence of morphogenetic cell movements, the coordination of which requires precise interpretation of signaling cues by heart primordia. Here we show that Nodal functions cooperatively with FGF during heart tube formation and asymmetric placement. Both pathways act as migratory stimuli for cardiac progenitor cells (CPCs), but FGF is dispensable for directing heart tube asymmetry, which is governed by Nodal. We further find that Nodal controls CPC migration by inducing left-right asymmetries in the formation of actin-based protrusions in CPCs. Additionally, we define a developmental window in which FGF signals are required for proper heart looping and show cooperativity between FGF and Nodal in this process. We present evidence FGF may promote heart looping through addition of the secondary heart field. Finally, we demonstrate that loss of FGF signaling affects proper development of the atrioventricular canal (AVC), which likely contributes to abnormal chamber morphologies in FGF-deficient hearts. Together, our data shed insight into how the spatiotemporal dynamics of signaling cues regulate the cellular behaviors underlying organ morphogenesis.

Efficient Genome Editing with Chimeric Oligonucleotide-Directed Editing.

Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report new nCas9-DNA polymerase fusion proteins to create chimeric oligonucleotide-directed editing (CODE) systems for search-and-replace genome editing. Through successive rounds of engineering, we developed CODEMax and CODEMax(exo+) editors that achieve efficient genome modifications in human cells with low unintended edits. CODEMax and CODEMax(exo+) contain an engineered Bst DNA polymerase derivative known for its robust strand displacement ability. Additionally, CODEMax(exo+) features a 5' to 3' exonuclease activity that promotes effective strand invasion and repair outcomes favoring the incorporation of the desired edit. We demonstrate CODEs can perform small insertions, deletions, and substitutions with improved efficiency compared to PEMax at many loci. Overall, CODEs complement existing prime editors to expand the toolbox for genome manipulations without double-stranded breaks.

A twist in Pitx2 regulation of gut looping.

Asymmetric expression of the transcription factor Pitx2 is important for correct asymmetry in organs during development. In a recent issue of Science, Sanketi et al. find Pitx2 expression directing gut tilting is independent of Nodal and acts as a "brake" to counteract BMP4 signaling on the right.

Promoting validation and cross-phylogenetic integration in model organism research.

Model organism (MO) research provides a basic understanding of biology and disease due to the evolutionary conservation of the molecular and cellular language of life. MOs have been used to identify and understand the function of orthologous genes, proteins, cells and tissues involved in biological processes, to develop and evaluate techniques and methods, and to perform whole-organism-based chemical screens to test drug efficacy and toxicity. However, a growing richness of datasets and the rising power of computation raise an important question: How do we maximize the value of MOs? In-depth discussions in over 50 virtual presentations organized by the National Institutes of Health across more than 10 weeks yielded important suggestions for improving the rigor, validation, reproducibility and translatability of MO research. The effort clarified challenges and opportunities for developing and integrating tools and resources. Maintenance of critical existing infrastructure and the implementation of suggested improvements will play important roles in maintaining productivity and facilitating the validation of animal models of human biology and disease.