Neurite outgrowth deficits caused by rare PLXNB1 mutation in pediatric bipolar disorder.

Pediatric bipolar disorder (PBD) is a severe mood dysregulation condition that affects 0.5-1% of children and teens in the United States. It is associated with recurrent episodes of mania and depression and an increased risk of suicidality. However, the genetics and neuropathology of PBD are largely unknown. Here, we used a combinatorial family-based approach to characterize cellular, molecular, genetic, and network-level deficits associated with PBD. We recruited a PBD patient and three unaffected family members from a family with a history of psychiatric illnesses. Using resting-state functional magnetic resonance imaging (rs-fMRI), we detected altered resting-state functional connectivity in the patient as compared to an unaffected sibling. Using transcriptomic profiling of patient and control induced pluripotent stem cell (iPSC)-derived telencephalic organoids, we found aberrant signaling in the molecular pathways related to neurite outgrowth. We corroborated the presence of neurite outgrowth deficits in patient iPSC-derived cortical neurons and identified a rare homozygous loss-of-function PLXNB1 variant (c.1360C>C; p.Ser454Arg) responsible for the deficits in the patient. Expression of wild-type PLXNB1, but not the variant, rescued neurite outgrowth in patient neurons, and expression of the variant caused the neurite outgrowth deficits in cortical neurons from PlxnB1 knockout mice. These results indicate that dysregulated PLXNB1 signaling may contribute to an increased risk of PBD and other mood dysregulation-related disorders by disrupting neurite outgrowth and functional brain connectivity. Overall, this study established and validated a novel family-based combinatorial approach for studying cellular and molecular deficits in psychiatric disorders and identified dysfunctional PLXNB1 signaling and neurite outgrowth as potential risk factors for PBD.

DIS3 Variants are Associated With Primary Ovarian Insufficiency: Importance of Transcription/Translation in Oogenesis.

CONTEXT: A genetic etiology accounts for the majority of unexplained primary ovarian insufficiency (POI). OBJECTIVE: We hypothesized a genetic cause of POI for a sister pair with primary amenorrhea. DESIGN: The study was an observational study. Subjects were recruited at an academic institution. SUBJECTS: Subjects were sisters with primary amenorrhea caused by POI and their parents. Additional subjects included women with POI analyzed previously (n = 291). Controls were recruited for health in old age or were from the 1000 Genomes Project (total n = 233). INTERVENTION: We performed whole exome sequencing, and data were analyzed using the Pedigree Variant Annotation, Analysis and Search Tool, which identifies genes harboring pathogenic variants in families. We performed functional studies in a Drosophila melanogaster model. MAIN OUTCOME: Genes with rare pathogenic variants were identified. RESULTS: The sisters carried compound heterozygous variants in DIS3. The sisters did not carry additional rare variants that were absent in publicly available datasets. DIS3 knockdown in the ovary of D. melanogaster resulted in lack of oocyte production and severe infertility. CONCLUSIONS: Compound heterozygous variants in highly conserved amino acids in DIS3 and failure of oocyte production in a functional model suggest that mutations in DIS3 cause POI. DIS3 is a 3' to 5' exoribonuclease that is the catalytic subunit of the exosome involved in RNA degradation and metabolism in the nucleus. The findings provide further evidence that mutations in genes important for transcription and translation are associated with POI.

A dominant negative ADIPOQ mutation in a diabetic family with renal disease, hypoadiponectinemia, and hyperceramidemia.

Adiponectin, encoded by ADIPOQ, is an insulin-sensitizing, anti-inflammatory, and renoprotective adipokine that activates receptors with intrinsic ceramidase activity. We identified a family harboring a 10-nucleotide deletion mutation in ADIPOQ that cosegregates with diabetes and end-stage renal disease. This mutation introduces a frameshift in exon 3, resulting in a premature termination codon that disrupts translation of adiponectin's globular domain. Subjects with the mutation had dramatically reduced circulating adiponectin and increased long-chain ceramides levels. Functional studies suggest that the mutated protein acts as a dominant negative through its interaction with non-mutated adiponectin, decreasing circulating adiponectin levels, and correlating with metabolic disease.

Causal and Candidate Gene Variants in a Large Cohort of Women With Primary Ovarian Insufficiency.

CONTEXT: A genetic etiology likely accounts for the majority of unexplained primary ovarian insufficiency (POI). OBJECTIVE: We hypothesized that heterozygous rare variants and variants in enhanced categories are associated with POI. DESIGN: The study was an observational study. SETTING: Subjects were recruited at academic institutions. PATIENTS: Subjects from Boston (n = 98), the National Institutes of Health and Washington University (n = 98), Pittsburgh (n = 20), Italy (n = 43), and France (n = 32) were diagnosed with POI (amenorrhea with an elevated follicle-stimulating hormone level). Controls were recruited for health in old age or were from the 1000 Genomes Project (total n = 233). INTERVENTION: We performed whole exome sequencing (WES), and data were analyzed using a rare variant scoring method and a Bayes factor-based framework for identifying genes harboring pathogenic variants. We performed functional studies on identified genes that were not previously implicated in POI in a D. melanogaster model. MAIN OUTCOME: Genes with rare pathogenic variants and gene sets with increased burden of deleterious variants were identified. RESULTS: Candidate heterozygous variants were identified in known genes and genes with functional evidence. Gene sets with increased burden of deleterious alleles included the categories transcription and translation, DNA damage and repair, meiosis and cell division. Variants were found in novel genes from the enhanced categories. Functional evidence supported 7 new risk genes for POI (USP36, VCP, WDR33, PIWIL3, NPM2, LLGL1, and BOD1L1). CONCLUSIONS: Candidate causative variants were identified through WES in women with POI. Aggregating clinical data and genetic risk with a categorical approach may expand the genetic architecture of heterozygous rare gene variants causing risk for POI.

Heart morphogenesis gene regulatory networks revealed by temporal expression analysis.

During embryogenesis the heart forms as a linear tube that then undergoes multiple simultaneous morphogenetic events to obtain its mature shape. To understand the gene regulatory networks (GRNs) driving this phase of heart development, during which many congenital heart disease malformations likely arise, we conducted an RNA-seq timecourse in zebrafish from 30 hpf to 72 hpf and identified 5861 genes with altered expression. We clustered the genes by temporal expression pattern, identified transcription factor binding motifs enriched in each cluster, and generated a model GRN for the major gene batteries in heart morphogenesis. This approach predicted hundreds of regulatory interactions and found batteries enriched in specific cell and tissue types, indicating that the approach can be used to narrow the search for novel genetic markers and regulatory interactions. Subsequent analyses confirmed the GRN using two mutants, Tbx5 and nkx2-5, and identified sets of duplicated zebrafish genes that do not show temporal subfunctionalization. This dataset provides an essential resource for future studies on the genetic/epigenetic pathways implicated in congenital heart defects and the mechanisms of cardiac transcriptional regulation.

The heparan sulfate editing enzyme Sulf1 plays a novel role in zebrafish VegfA mediated arterial venous identity.

Arterial and venous specification is critical for establishing and maintaining a functioning vascular system, and defects in key arteriovenous signaling pathways including VEGF (vascular endothelial growth factor) lead to congenital arteriopathies. The activities of VEGF, are in part controlled by heparan sulfate (HS) proteoglycans, significant components of the endothelial glycocalyx. The level of 6-O sulfation on HS polysaccharide chains, that mediate the interaction between HS and VEGFA, is edited at the cell surface by the enzyme SULF1. We investigated the role of sulf1 in vascular development. In zebrafish sulf1 is expressed in the head and tail vasculature, corresponding spatially and temporally with vascular development. Targeted knockdown of sulf1 by antisense morpholinos resulted in severe vascular patterning and maturation defects. 93 % of sulf1 morphants show dysmorphogenesis in arterial development leading to occlusion of the distal aorta and lack of axial and cranial circulation. Co-injection of vegfa165 mRNA rescued circulatory defects. While the genes affecting haematopoiesis are unchanged, expression of several arterial markers downstream of VegfA signalling such as notch and ephrinB2 are severely reduced in the dorsal aorta, with a concomitant increase in expression of the venous markers flt4 in the dorsal aorta of the morphants. Furthermore, in vitro, lack of SULF1 expression downregulates VEGFA-mediated arterial marker expression, confirming that Sulf1 mediates arterial specification by regulating VegfA165 activity. This study provides the first in vivo evidence for the integral role of the endothelial glycocalyx in specifying arterial-venous identity, vascular patterning and arterial integrity, and will help to better understand congenital arteriopathies.

MMAPPR: mutation mapping analysis pipeline for pooled RNA-seq.

Forward genetic screens in model organisms are vital for identifying novel genes essential for developmental or disease processes. One drawback of these screens is the labor-intensive and sometimes inconclusive process of mapping the causative mutation. To leverage high-throughput techniques to improve this mapping process, we have developed a Mutation Mapping Analysis Pipeline for Pooled RNA-seq (MMAPPR) that works without parental strain information or requiring a preexisting SNP map of the organism, and adapts to differential recombination frequencies across the genome. MMAPPR accommodates the considerable amount of noise in RNA-seq data sets, calculates allelic frequency by Euclidean distance followed by Loess regression analysis, identifies the region where the mutation lies, and generates a list of putative coding region mutations in the linked genomic segment. MMAPPR can exploit RNA-seq data sets from isolated tissues or whole organisms that are used for gene expression and transcriptome analysis in novel mutants. We tested MMAPPR on two known mutant lines in zebrafish, nkx2.5 and tbx1, and used it to map two novel ENU-induced cardiovascular mutants, with mutations found in the ctr9 and cds2 genes. MMAPPR can be directly applied to other model organisms, such as Drosophila and Caenorhabditis elegans, that are amenable to both forward genetic screens and pooled RNA-seq experiments. Thus, MMAPPR is a rapid, cost-efficient, and highly automated pipeline, available to perform mutant mapping in any organism with a well-assembled genome.

Dynamic expression patterns of 6-O endosulfatases during zebrafish development suggest a subfunctionalisation event for sulf2.

The 6-O-endosulfatase enzymes (Sulfs) edit the final sulfation pattern and function of heparan sulfate (HS) by removal of 6-O-sulfate groups from the chain. To date, two mammalian sulf genes have been identified that regulate many signalling pathways during embryonic development. In zebrafish a sulf1 ortholog and duplicate copies of the mammalian sulf2 gene, sulf2a and sulf2, have been identified, which contain conserved motifs characteristic of vertebrate sulf genes. Zebrafish sulf1 and sulf2a are broadly expressed in the central nervous system (CNS) and non-neuronal tissue including heart, somite boundaries, olfactory system, and otic vesicle, whereas sulf2 expression is almost entirely restricted to the CNS. The duplicate copies of sulf2 have distinct expression patterns, which together mirror that of mouse sulf2, suggesting duplication in the teleost lineage has been followed by subfunctionalisation, whereby both genes need to be preserved by selection to ensure the ancestral gene's expression profile and function is maintained.

Tinkering with heparan sulfate sulfation to steer development.

Heparan sulfate (HS) proteoglycans, at the cell surface and extracellular matrix, facilitate ligand-receptor interactions crucial to many physiological processes. The distinct sulfation patterns of HS sugar chains presented by their protein core are key to HS proteoglycan activity. Tight regulation of several Golgi complex enzyme families is crucial to produce complex tissue-specific HS sequences. Several in vivo models deficient in HS biosynthesis enzymes demonstrate that developmental abnormalities result from modified HS structure. This review will discuss the plasticity of sulfation requirements on HS for activating protein ligands, which might reflect a flexible HS biosynthetic mechanism. In addition, the latest discovery of HS acting enzymes, the Sulfs, responsible for extracellular tweaking of HS sulfation levels subsequent to biosynthesis will be considered.