Duplicated zebrafish (Danio rerio) inositol phosphatases inpp5ka and inpp5kb diverged in expression pattern and function.

One hurdle in the development of zebrafish models of human disease is the presence of multiple zebrafish orthologs resulting from whole genome duplication in teleosts. Mutations in inositol polyphosphate 5-phosphatase K (INPP5K) lead to a syndrome characterized by variable presentation of intellectual disability, brain abnormalities, cataracts, muscle disease, and short stature. INPP5K is a phosphatase acting at position 5 of phosphoinositides to control their homeostasis and is involved in insulin signaling, cytoskeletal regulation, and protein trafficking. Previously, our group and others have replicated the human phenotypes in zebrafish knockdown models by targeting both INPP5K orthologs inpp5ka and inpp5kb. Here, we show that inpp5ka is the more closely related orthologue to human INPP5K. While both inpp5ka and inpp5kb mRNA expression levels follow a similar trend in the developing head, eyes, and tail, inpp5ka is much more abundantly expressed in these tissues than inpp5kb. In situ hybridization revealed a similar trend, also showing unique localization of inpp5kb in the pineal gland and retina indicating different transcriptional regulation. We also found that inpp5kb has lost its catalytic activity against its preferred substrate, PtdIns(4,5)P2. Since most human mutations are missense changes disrupting phosphatase activity, we propose that loss of inpp5ka alone can be targeted to recapitulate the human presentation. In addition, we show that the function of inpp5kb has diverged from inpp5ka and may play a novel role in the zebrafish.

Modeling and integration of N-glycan biomarkers in a comprehensive biomarker data model.

Molecular biomarkers measure discrete components of biological processes that can contribute to disorders when impaired. Great interest exists in discovering early cancer biomarkers to improve outcomes. Biomarkers represented in a standardized data model, integrated with multi-omics data, may improve the understanding and use of novel biomarkers such as glycans and glycoconjugates. Among altered components in tumorigenesis, N-glycans exhibit substantial biomarker potential, when analyzed with their protein carriers. However, such data are distributed across publications and databases of diverse formats, which hamper their use in research and clinical application. Mass spectrometry measures of 50 N-glycans on 7 serum proteins in liver disease were integrated (as a panel) into a cancer biomarker data model, providing a unique identifier, standard nomenclature, links to glycan resources, and accession and ontology annotations to standard protein, gene, disease, and biomarker information. Data provenance was documented with a standardized United States Food and Drug Administration-supported BioCompute Object. Using the biomarker data model allows the capture of granular information, such as glycans with different levels of abundance in cirrhosis, hepatocellular carcinoma, and transplant groups. Such representation in a standardized data model harmonizes glycomics data in a unified framework, making glycan-protein biomarker data exploration more available to investigators and to other data resources. The biomarker data model we describe can be used by researchers to describe their novel glycan and glycoconjugate biomarkers; it can integrate N-glycan biomarker data with multi-source biomedical data and can foster discovery and insight within a unified data framework for glycan biomarker representation, thereby making the data FAIR (Findable, Accessible, Interoperable, Reusable) (https://www.go-fair.org/fair-principles/).

Genomics of Black American colon cancer disparities: An RNA sequencing (RNA-Seq) study from an academic, tertiary referral center.

BACKGROUND: Black Americans have a higher incidence and mortality rate from colorectal cancer compared to their non-Hispanic White American counterparts. Even when controlling for sociodemographic differences between these 2 populations, Black Americans remain disproportionately affected by colorectal cancer. The purpose of our study was to determine if differences in gene expression between Black American and non-Hispanic White American colon cancer specimens could help explain differences in the incidence and mortality rate between these 2 populations. METHODS: Black Americans and non-Hispanic White Americans undergoing colon resection for stages I, II, or III colon cancer at a single institution were identified. Black American and non-Hispanic White American patients were matched for age, sex, and colon cancer stage to minimize the risk of confounding variables. Tissue samples were obtained at the time of colon resection and were analyzed using RNA sequencing to determine if there were differences in the expression of genes and biologic processes between the 2 groups. RESULTS: A total of 17 colon cancer specimens were analyzed; 8 (47.1%) patients were Black Americans. A total of 456 genes were identified as being expressed differently (ie, up or downregulated) in Black American compared to non-Hispanic White American colon cancer specimens. Moreover, 500 different genetic pathways were noted to be significantly over-represented with differentially expressed genes in our comparison of Black American and non-Hispanic White American colon cancer specimens, the majority of which plays a role in inflammation and immune cell function. CONCLUSION: Significant differences in gene expression and genetic pathways exist between Black Americans and non-Hispanic White Americans. Additional and multi-institutional and registry-based studies are needed to validate our findings and to further elucidate the contribution that these differences have to the overall incidence and mortality rate from colon cancer in these 2 patient populations.

COVID-19 biomarkers and their overlap with comorbidities in a disease biomarker data model.

In response to the COVID-19 outbreak, scientists and medical researchers are capturing a wide range of host responses, symptoms and lingering postrecovery problems within the human population. These variable clinical manifestations suggest differences in influential factors, such as innate and adaptive host immunity, existing or underlying health conditions, comorbidities, genetics and other factors-compounding the complexity of COVID-19 pathobiology and potential biomarkers associated with the disease, as they become available. The heterogeneous data pose challenges for efficient extrapolation of information into clinical applications. We have curated 145 COVID-19 biomarkers by developing a novel cross-cutting disease biomarker data model that allows integration and evaluation of biomarkers in patients with comorbidities. Most biomarkers are related to the immune (SAA, TNF-∝ and IP-10) or coagulation (D-dimer, antithrombin and VWF) cascades, suggesting complex vascular pathobiology of the disease. Furthermore, we observe commonality with established cancer biomarkers (ACE2, IL-6, IL-4 and IL-2) as well as biomarkers for metabolic syndrome and diabetes (CRP, NLR and LDL). We explore these trends as we put forth a COVID-19 biomarker resource (https://data.oncomx.org/covid19) that will help researchers and diagnosticians alike.

Novel mutation identification and copy number variant detection via exome sequencing in congenital muscular dystrophy.

BACKGROUND: Congenital muscular dystrophy type 1A (MDC1A), also termed merosin-deficient congenital muscular dystrophy (CMD), is a severe form of CMD caused by mutations in the laminin α2 gene (LAMA2). Of the more than 300 likely pathogenic variants found in the Leiden Open Variant Database, the majority are truncating mutations leading to complete LAMA2 loss of function, but multiple copy number variants (CNVs) have also been reported with variable frequency. METHODS: We collected a cohort of individuals diagnosed with likely MDC1A and sought to identify both single nucleotide variants and small and larger CNVs via exome sequencing by extending the analysis of sequencing data to detect splicing changes and CNVs. RESULTS: Standard exome analysis identified multiple novel LAMA2 variants in our cohort, but only four cases carried biallelic variants. Since likely truncating LAMA2 variants are often found in heterozygosity without a second allele, we performed additional splicing and CNV analysis on exome data and identified one splice change outside of the canonical sequences and three CNVs, in the remaining four cases. CONCLUSIONS: Our findings support the expectation that a portion of MDC1A cases may be caused by at least one CNV allele and show how these changes can be effectively identified by additional analysis of existing exome data.

Myopathic changes associated with psychomotor delay and seizures caused by a novel homozygous mutation in TBCK.

BACKGROUND: Biallelic mutations in TBC1-domain containing kinase (TBCK) lead to hypotonia, global developmental delay with severe cognitive and motor deficits, and variable presentation of dysmorphic facial features and brain malformations. It remains unclear whether hypotonia in these individuals is purely neurogenic, or also caused by progressive muscle disease. METHODS: Whole exome sequencing was performed on a family diagnosed with nonspecific myopathic changes by means of histological analysis and immunohistochemistry of muscle biopsy samples. RESULTS: A novel homozygous truncation in TBCK was found in two sisters diagnosed with muscle disease and severe psychomotor delay. TBCK was completely absent in these patients. CONCLUSIONS: Our findings identify a novel early truncating variant in TBCK associated with a severe presentation and add muscle disease to the variability of phenotypes associated with TBCK mutations. Inconsistent genotype/phenotype correlation could be ascribed to the multiple roles of TBCK in intracellular signaling and endolysosomal function in different tissues.

Overlap of polymicrogyria, hydrocephalus, and Joubert syndrome in a family with novel truncating mutations in ADGRG1/GPR56 and KIAA0556.

Genetic mutations associated with brain malformations can lead to a spectrum of severity and it is often difficult to determine whether there are additional pathogenic variants contributing to the phenotype. Here, we present a family affected by a severe brain malformation including bilateral polymicrogyria, hydrocephalus, patchy white matter signal changes, and cerebellar and pontine hypoplasia with elongated cerebellar peduncles leading to the molar tooth sign. While the malformation is reminiscent of bilateral frontoparietal polymicrogyria (BFPP), the phenotype is more severe than previously reported and also includes features of Joubert syndrome (JBTS). Via exome sequencing, we identified homozygous truncating mutations in both ADGRG1/GPR56 and KIAA0556, which are known to cause BFPP and mild brain-specific JBTS, respectively. This study shows how two independent mutations can interact leading to complex brain malformations.

De novo and biallelic DEAF1 variants cause a phenotypic spectrum.

PURPOSE: To investigate the effect of different DEAF1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and on DEAF1 activity in vitro. METHODS: We assembled a cohort of 23 patients with de novo and biallelic DEAF1 variants, described the genotype-phenotype correlation, and investigated the differential effect of de novo and recessive variants on transcription assays using DEAF1 and Eif4g3 promoter luciferase constructs. RESULTS: The proportion of the most prevalent phenotypic features, including intellectual disability, speech delay, motor delay, autism, sleep disturbances, and a high pain threshold, were not significantly different in patients with biallelic and pathogenic de novo DEAF1 variants. However, microcephaly was exclusively observed in patients with recessive variants (p < 0.0001). CONCLUSION: We propose that different variants in the DEAF1 gene result in a phenotypic spectrum centered around neurodevelopmental delay. While a pathogenic de novo dominant variant would also incapacitate the product of the wild-type allele and result in a dominant-negative effect, a combination of two recessive variants would result in a partial loss of function. Because the clinical picture can be nonspecific, detailed phenotype information, segregation, and functional analysis are fundamental to determine the pathogenicity of novel variants and to improve the care of these patients.

NMDA receptors potentiate activity-dependent dendritic release of neuropeptides from hypothalamic neurons.

KEY POINTS: Using 'sniffer' cell biosensors, we evaluated the effects of specific firing patterns and frequencies on activity-dependent somatodendritic release of vasopressin from paraventricular nucleus neurones. Somatodendritic release of vasopressin was rarely observed during continuous firing but was strengthened by clustered activity. Moreover, release evoked at any given frequency was robustly potentiated by NMDA receptor (NMDAR)-mediated firing. Differently from axonal release, NMDAR activation was necessary for somatodendritic release to occur at physiological firing frequencies, acting thus as a gating mechanism by which activity-dependent release from these two neuronal compartments could be independently regulated. The NMDAR-mediated potentiation was independent of a specific firing pattern and was not accompanied by increased spike broadening, but correlated with higher dendritic Ca2+ levels. Our studies provide fundamental novel information regarding stimulus-secretion coupling at somatodendritic compartments, and shed light into mechanisms by which activity-dependent release of neuronal signals from axonal terminals and dendrites could be regulated in a spatially compartmentalized manner. ABSTRACT: Dendrites are now recognized to be active transmitting neuronal compartments subserving complex brain functions, including motor behaviours and homeostatic neurohumoral responses. Still, the precise mechanisms underlying activity-dependent release of dendritic signals, and how dendritic release is regulated independently from axonal release, remains largely unknown. We used 'sniffer' biosensor cells to enable the measurement and study of activity-dependent dendritic release of vasopressin (VP) from hypothalamic neurones in brain slices. SnifferVP responses were dose-dependent, with a threshold detection level of 0.5 nM for VP, being thus a highly sensitive tool to detect endogenous physiological levels of the neuropeptide. Somatodendritic release of VP was rarely observed in response to a burst of action potentials fired in continuous mode, but was strengthened by clustered firing activity. Moreover, release evoked at any given frequency was robustly potentiated when firing was triggered by NMDA receptor (NMDAR) activation. Differently from axonal release, NMDAR activation was necessary for dendritic release to occur at physiological firing frequencies. Thus, we propose that NMDARs may act as a gating mechanism by which activity-dependent release from these two neuronal compartments can be independently regulated. The NMDAR-mediated potentiation of dendritic release was independent of a particular action potential waveform, firing pattern evoked, or a more pronounced spiked broadening, but correlated with higher dendritic Ca2+ levels. Overall, our studies provide fundamental novel information regarding stimulus-secretion coupling at neuronal dendrites, and shed light into mechanisms by which activity-dependent release of neuronal signals from axonal terminals and dendrites can be regulated in a spatially compartmentalized manner.

Chronic activation of hypothalamic oxytocin neurons improves cardiac function during left ventricular hypertrophy-induced heart failure.

AIMS: A distinctive hallmark of heart failure (HF) is autonomic imbalance, consisting of increased sympathetic activity, and decreased parasympathetic tone. Recent work suggests that activation of hypothalamic oxytocin (OXT) neurons could improve autonomic balance during HF. We hypothesized that a novel method of chronic selective activation of hypothalamic OXT neurons will improve cardiac function and reduce inflammation and fibrosis in a rat model of HF. METHODS AND RESULTS: Two groups of male Sprague-Dawley rats underwent trans-ascending aortic constriction (TAC) to induce left ventricular (LV) hypertrophy that progresses to HF. In one TAC group, OXT neurons in the paraventricular nucleus of the hypothalamus were chronically activated by selective expression and activation of excitatory DREADDs receptors with daily injections of clozapine N-oxide (CNO) (TAC + OXT). Two additional age-matched groups received either saline injections (Control) or CNO injections for excitatory DREADDs activation (OXT NORM). Heart rate (HR), LV developed pressure (LVDP), and coronary flow rate were measured in isolated heart experiments. Isoproterenol (0.01 nM-1.0 µM) was administered to evaluate ß-adrenergic sensitivity. We found that increases in cellular hypertrophy and myocardial collagen density in TAC were blunted in TAC + OXT animals. Inflammatory cytokine IL-1ß expression was more than twice higher in TAC than all other hearts. LVDP, rate pressure product (RPP), contractility, and relaxation were depressed in TAC compared with all other groups. The response of TAC and TAC + OXT hearts to isoproterenol was blunted, with no significant increase in RPP, contractility, or relaxation. However, HR in TAC + OXT animals increased to match Control at higher doses of isoproterenol. CONCLUSIONS: Activation of hypothalamic OXT neurons to elevate parasympathetic tone reduced cellular hypertrophy, levels of IL-1ß, and fibrosis during TAC-induced HF in rats. Cardiac contractility parameters were significantly higher in TAC + OXT compared with TAC animals. HR sensitivity, but not contractile sensitivity, to ß-adrenergic stimulation was improved in TAC + OXT hearts.

Mutations in INPP5K Cause a Form of Congenital Muscular Dystrophy Overlapping Marinesco-Sjögren Syndrome and Dystroglycanopathy.

Congenital muscular dystrophies display a wide phenotypic and genetic heterogeneity. The combination of clinical, biochemical, and molecular genetic findings must be considered to obtain the precise diagnosis and provide appropriate genetic counselling. Here we report five individuals from four families presenting with variable clinical features including muscular dystrophy with a reduction in dystroglycan glycosylation, short stature, intellectual disability, and cataracts, overlapping both the dystroglycanopathies and Marinesco-Sjögren syndrome. Whole-exome sequencing revealed homozygous missense and compound heterozygous mutations in INPP5K in the affected members of each family. INPP5K encodes the inositol polyphosphate-5-phosphatase K, also known as SKIP (skeletal muscle and kidney enriched inositol phosphatase), which is highly expressed in the brain and muscle. INPP5K localizes to both the endoplasmic reticulum and to actin ruffles in the cytoplasm. It has been shown to regulate myoblast differentiation and has also been implicated in protein processing through its interaction with the ER chaperone HSPA5/BiP. We show that morpholino-mediated inpp5k loss of function in the zebrafish results in shortened body axis, microphthalmia with disorganized lens, microcephaly, reduced touch-evoked motility, and highly disorganized myofibers. Altogether these data demonstrate that mutations in INPP5K cause a congenital muscular dystrophy syndrome with short stature, cataracts, and intellectual disability.

RNA2DNAlign: nucleotide resolution allele asymmetries through quantitative assessment of RNA and DNA paired sequencing data.

We introduce RNA2DNAlign, a computational framework for quantitative assessment of allele counts across paired RNA and DNA sequencing datasets. RNA2DNAlign is based on quantitation of the relative abundance of variant and reference read counts, followed by binomial tests for genotype and allelic status at SNV positions between compatible sequences. RNA2DNAlign detects positions with differential allele distribution, suggesting asymmetries due to regulatory/structural events. Based on the type of asymmetry, RNA2DNAlign outlines positions likely to be implicated in RNA editing, allele-specific expression or loss, somatic mutagenesis or loss-of-heterozygosity (the first three also in a tumor-specific setting). We applied RNA2DNAlign on 360 matching normal and tumor exomes and transcriptomes from 90 breast cancer patients from TCGA. Under high-confidence settings, RNA2DNAlign identified 2038 distinct SNV sites associated with one of the aforementioned asymetries, the majority of which have not been linked to functionality before. The performance assessment shows very high specificity and sensitivity, due to the corroboration of signals across multiple matching datasets. RNA2DNAlign is freely available from http://github.com/HorvathLab/NGS as a self-contained binary package for 64-bit Linux systems.

Neurotransmission to parasympathetic cardiac vagal neurons in the brain stem is altered with left ventricular hypertrophy-induced heart failure.

Hypertension, cardiac hypertrophy, and heart failure (HF) are widespread and debilitating cardiovascular diseases that affect nearly 23 million people worldwide. A distinctive hallmark of these cardiovascular diseases is autonomic imbalance, with increased sympathetic activity and decreased parasympathetic vagal tone. Recent device-based approaches, such as implantable vagal stimulators that stimulate a multitude of visceral sensory and motor fibers in the vagus nerve, are being evaluated as new therapeutic approaches for these and other diseases. However, little is known about how parasympathetic activity to the heart is altered with these diseases, and this lack of knowledge is an obstacle in the goal of devising selective interventions that can target and selectively restore parasympathetic activity to the heart. To identify the changes that occur within the brain stem to diminish the parasympathetic cardiac activity, left ventricular hypertrophy was elicited in rats by aortic pressure overload using a transaortic constriction approach. Cardiac vagal neurons (CVNs) in the brain stem that generate parasympathetic activity to the heart were identified with a retrograde tracer and studied using patch-clamp electrophysiological recordings in vitro. Animals with left cardiac hypertrophy had diminished excitation of CVNs, which was mediated both by an augmented frequency of spontaneous inhibitory GABAergic neurotransmission (with no alteration of inhibitory glycinergic activity) as well as a diminished amplitude and frequency of excitatory neurotransmission to CVNs. Opportunities to alter these network pathways and neurotransmitter receptors provide future targets of intervention in the goal to restore parasympathetic activity and autonomic balance to the heart in cardiac hypertrophy and other cardiovascular diseases.