Rare homozygous cilia gene variants identified in consanguineous congenital heart disease patients.

Congenital heart defects (CHD) appear in almost one percent of live births. Asian countries have the highest birth prevalence of CHD in the world. Recessive genotypes may represent a CHD risk factor in Asian populations with a high degree of consanguineous marriages. Genetic analysis of consanguineous families may represent a relatively unexplored source for investigating CHD etiology. To obtain insight into the contribution of recessive genotypes in CHD we analysed a cohort of forty-nine Pakistani CHD probands, originating from consanguineous unions. The majority (82%) of patient's malformations were septal defects. We identified protein altering, rare homozygous variants (RHVs) in the patient's coding genome by whole exome sequencing. The patients had a median of seven damaging RHVs each, and our analysis revealed a total of 758 RHVs in 693 different genes. By prioritizing these genes based on variant severity, loss-of-function intolerance and specific expression in the developing heart, we identified a set of 23 candidate disease genes. These candidate genes were significantly enriched for genes known to cause heart defects in recessive mouse models (P < 2.4e-06). In addition, we found a significant enrichment of cilia genes in both the initial set of 693 genes (P < 5.4e-04) and the 23 candidate disease genes (P < 5.2e-04). Functional investigation of ADCY6 in cell- and zebrafish-models verified its role in heart development. Our results confirm a significant role for cilia genes in recessive forms of CHD and suggest important functions of cilia genes in cardiac septation.

α3Na+/K+-ATPase deficiency causes brain ventricle dilation and abrupt embryonic motility in zebrafish.

Na(+)/K(+)-ATPases are transmembrane ion pumps that maintain ion gradients across the basolateral plasma membrane in all animal cells to facilitate essential biological functions. Mutations in the Na(+)/K(+)-ATPase α3 subunit gene (ATP1A3) cause rapid-onset dystonia-parkinsonism, a rare movement disorder characterized by sudden onset of dystonic spasms and slow movements. In the brain, ATP1A3 is principally expressed in neurons. In zebrafish, the transcripts of the two ATP1A3 orthologs, Atp1a3a and Atp1a3b, show distinct expression in the brain. Surprisingly, targeted knockdown of either Atp1a3a or Atp1a3b leads to brain ventricle dilation, a likely consequence of ion imbalances across the plasma membrane that cause accumulation of cerebrospinal fluid in the ventricle. The brain ventricle dilation is accompanied by a depolarization of spinal Rohon-Beard neurons in Atp1a3a knockdown embryos, suggesting impaired neuronal excitability. This is further supported by Atp1a3a or Atp1a3b knockdown results where altered responses to tactile stimuli as well as abnormal motility were observed. Finally, proteomic analysis identified several protein candidates highlighting proteome changes associated with the knockdown of Atp1a3a or Atp1a3b. Our data thus strongly support the role of α3Na(+)/K(+)-ATPase in zebrafish motility and brain development, associating for the first time the α3Na(+)/K(+)-ATPase deficiency with brain ventricle dilation.

The α2Na+/K+-ATPase is critical for skeletal and heart muscle function in zebrafish.

The Na(+)/K(+)-ATPase generates ion gradients across the plasma membrane, essential for multiple cellular functions. In mammals, four different Na(+)/K(+)-ATPase α-subunit isoforms are associated with characteristic cell-type expression profiles and kinetics. We found the zebrafish α2Na(+)/K(+)-ATPase associated with striated muscles and that knockdown causes a significant depolarization of the resting membrane potential in slow-twitch fibers of skeletal muscles. Abrupt mechanosensory responses were observed in α2Na(+)/K(+)-ATPase-deficient embryos, possibly linked to a postsynaptic defect. The α2Na(+)/K(+)-ATPase deficiency reduced the heart rate and caused a loss of left-right asymmetry in the heart tube. Similar phenotypes from knockdown of the Na(+)/Ca(2+) exchanger indicated a role for the interplay between these two proteins in the observed phenotypes. Furthermore, proteomics identified up- and downregulation of specific phenotype-related proteins, such as parvalbumin, CaM, GFAP and multiple kinases, thus highlighting a potential proteome change associated with the dynamics of α2Na(+)/K(+)-ATPase. Taken together, our findings show that zebrafish α2Na(+)/K(+)-ATPase is important for skeletal and heart muscle functions.

Systems genetics analysis identifies calcium-signaling defects as novel cause of congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) occurs in almost 1% of newborn children and is considered a multifactorial disorder. CHD may segregate in families due to significant contribution of genetic factors in the disease etiology. The aim of the study was to identify pathophysiological mechanisms in families segregating CHD. METHODS: We used whole exome sequencing to identify rare genetic variants in ninety consenting participants from 32 Danish families with recurrent CHD. We applied a systems biology approach to identify developmental mechanisms influenced by accumulation of rare variants. We used an independent cohort of 714 CHD cases and 4922 controls for replication and performed functional investigations using zebrafish as in vivo model. RESULTS: We identified 1785 genes, in which rare alleles were shared between affected individuals within a family. These genes were enriched for known cardiac developmental genes, and 218 of these genes were mutated in more than one family. Our analysis revealed a functional cluster, enriched for proteins with a known participation in calcium signaling. Replication in an independent cohort confirmed increased mutation burden of calcium-signaling genes in CHD patients. Functional investigation of zebrafish orthologues of ITPR1, PLCB2, and ADCY2 verified a role in cardiac development and suggests a combinatorial effect of inactivation of these genes. CONCLUSIONS: The study identifies abnormal calcium signaling as a novel pathophysiological mechanism in human CHD and confirms the complex genetic architecture underlying CHD.

RRP7A links primary microcephaly to dysfunction of ribosome biogenesis, resorption of primary cilia, and neurogenesis.

Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.

CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-ß/BMP Signaling at the Primary Cilium.

The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure but caused impaired transforming growth factor-ß/bone morphogenetic protein (TGF-ß/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss of CEP128, and quantitative phosphoproteomics revealed that CEP128 loss affects TGF-ß1-induced phosphorylation of multiple proteins that regulate cilium-associated vesicle trafficking.

Assay for Transposase-Accessible Chromatin with High-Throughput Sequencing (ATAC-Seq) Protocol for Zebrafish Embryos.

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a useful method to map genome-wide chromatin accessibility and nucleosome positioning. Genome-wide sequencing is performed utilizing adapter sequences inserted by a prokaryotic transposase, Tn5, into the accessible regions of chromatin. Here we describe the use of ATAC-seq in the zebrafish embryo and thereby the applicability of this approach in whole vertebrate embryos.

Zebrafish Whole-Mount In Situ Hybridization Followed by Sectioning.

In situ hybridization is a powerful technique used for locating specific nucleic acid targets within morphologically preserved tissues and cell preparations. A labeled RNA or DNA probe hybridizes to its complementary mRNA or DNA sequence within a sample. Here, we describe RNA in situ hybridization protocol for whole-mount zebrafish embryos.

Whole-Mount Immunohistochemistry for Anti-F59 in Zebrafish Embryos (1-5 Days Post Fertilization (dpf)).

Immunohistochemistry (IHC) is a powerful method to determine localization of tissue components by the interaction of target antigens with labeled antibodies. Here we describe an IHC protocol for localizing the myosin heavy chain of zebrafish embryos at 1-2 and 3-5 days post fertilization (dpf).

Zebrafish as a novel model to assess Na+/K(+)-ATPase-related neurological disorders.

Modeling neurological disorders using zebrafish increases rapidly as this model system allows easy access to all developmental stages and imaging of pathological processes. A surprising degree of functional conservation has been demonstrated between human genes implicated in neurodegenerative diseases and their zebrafish orthologues. Zebrafish offers rapid high throughput screening of therapeutic compounds and live imaging of pathogenic mechanisms in vivo. Several recent zebrafish studies functionally assessed the role of the sodium-potassium pump (Na(+)/K(+)-ATPase). The Na(+)/K(+)-ATPase maintains the electrochemical gradients across the plasma membrane, essential for e.g. signaling, secondary active transport, glutamate re-uptake and neuron excitability in animal cells. Na(+)/K(+)-ATPase mutations are associated with neurological disorders, where mutations in the Na(+)/K(+)-ATPase α2 and α3 isoforms cause Familial hemiplegic migraine type 2 (FHM2) and Rapid-onset dystonia-parkinsonism (RDP)/Alternating hemiplegic childhood (AHC), respectively. In zebrafish, knock-down of Na(+)/K(+)-ATPase isoforms included skeletal and heart muscle defects, impaired embryonic motility, depolarized Rohon-beard neurons and abrupt brain ventricle development. In this review, we discuss zebrafish as a model to assess Na(+)/K(+)-ATPase isoform functions. Furthermore, studies investigating proteomic changes in both α2- and α3-isoform deficient embryos and their potential connections to the Na(+)/K(+)-ATPase functions will be discussed.

Migraine- and dystonia-related disease-mutations of Na+/K+-ATPases: relevance of behavioral studies in mice to disease symptoms and neurological manifestations in humans.

The two autosomal dominantly inherited neurological diseases: familial hemiplegic migraine type 2 (FHM2) and familial rapid-onset of dystonia-parkinsonism (Familial RDP) are caused by in vivo mutations of specific alpha subunits of the sodium-potassium pump (Na(+)/K(+)-ATPase). Intriguingly, patients with classical FHM2 and RDP symptoms additionally suffer from other manifestations, such as epilepsy/seizures and developmental disabilities. Recent studies of FHM2 and RDP mouse models provide valuable tools for dissecting the vital roles of the Na(+)/K(+)-ATPases, and we discuss their relevance to the complex patient symptoms and manifestations. Thus, it is interesting that mouse models targeting a specific α-isoform cause different, although still comparable, phenotypes consistent with classical symptoms and other manifestations observed in FHM2 and RDP patients. This review highlights that use of mouse models have broad potentials for future research concerning migraine and dystonia-related diseases, which will contribute towards understanding the, yet unknown, pathophysiologies.

Early developmental expression of Mus musculus zinc finger RNA-binding protein compared to orthologs in Caenorhabditis elegans and Danio rerio and subcellular localization of Mus musculus and Caenorhabditis elegans zinc finger RNA-binding protein in 2-cell Mus musculus embryos.

In mouse, knock-out of the Zfr gene encoding the zinc finger RNA-binding protein (ZFR) is associated with early lethality during gastrulation, suggesting that a pool of maternally contributed Zfr mRNA might compensate to allow development. ZFR is an ancient and highly conserved chromosome-associated protein from nematodes to mammals. We characterized expression of the Zfr transcript during early development in Mus musculus, Danio rerio, and Caenorhabditis elegans by quantitative real-time polymerase chain reaction. Mouse Zfr mRNA was detected in all stages tested during mouse preimplantation, with higher levels at the 1-cell stage that includes the maternal contribution of Zfr mRNA. In D. rerio, Zfr mRNA expression was highest in unfertilized eggs and declines throughout development. In C. elegans, Zfr mRNA expression was barely detectable in the fertilized egg and the L1 stage, but increased in the adult organism. Microinjections of green fluorescent protein (GFP)-tagged versions of in vitro-transcribed mouse and C. elegans Zfr mRNAs into early mouse embryos allowed analysis of the intracellular localization of the protein. Mouse ZFR-GFP was localized in the nucleus in 2-cell stage embryos although absent from nucleoli. Deletion studies revealed that this nuclear localization required the C-terminal part of ZFR, as deletion of the C-terminal resulted in the localization to the nuclear membrane. Despite the lack of a conserved nuclear localization signal, the C. elegans ZFR-GFP fusion protein also displayed an intranuclear localization in the 2-cell mouse embryo.