The severity of MUSK pathogenic variants is predicted by the protein domain they disrupt.

Biallelic loss-of-function variants in the MUSK gene result in two allelic disorders: (1) congenital myasthenic syndrome (CMS; OMIM: 616325), a neuromuscular disorder that has a range of severity from severe neonatal-onset weakness to mild adult-onset weakness, and (2) fetal akinesia deformation sequence (OMIM: 208150), a form of pregnancy loss characterized by severe muscle weakness in the fetus. The MUSK gene codes for muscle-specific kinase (MuSK), a receptor tyrosine kinase involved in the development of the neuromuscular junction. Here, we report a case of neonatal-onset MUSK-related CMS in a patient harboring compound heterozygous deletions in the MUSK gene, including (1) a deletion of exons 2-3 leading to an in-frame MuSK protein lacking the immunoglobulin 1 (Ig1) domain and (2) a deletion of exons 7-11 leading to an out-of-frame, truncated MuSK protein. Individual domains of the MuSK protein have been elucidated structurally; however, a complete MuSK structure generated by machine learning algorithms has clear inaccuracies. We modify a predicted AlphaFold structure and integrate previously reported domain-specific structural data to suggest a MuSK protein that dimerizes in two locations (Ig1 and the transmembrane domain). We analyze known pathogenic variants in MUSK to discover domain-specific genotype-phenotype correlations; variants that lead to a loss of protein expression, disruption of the Ig1 domain, or Dok-7 binding are associated with the most severe phenotypes. A conceptual model is provided to explain the severe phenotypes seen in Ig1 variants and the poor response of our patient to pyridostigmine.

Specific CaMKIIs mediate convergent extension cell movements in early zebrafish development.

BACKGROUND: Noncanonical Wnts are morphogens that can elevate intracellular Ca2+, activate the Ca2+/calmodulin-dependent protein kinase, CaMKII, and promote cell movements during vertebrate gastrulation. RESULTS: Zebrafish express seven CaMKII genes during embryogenesis; two of these, camk2b1 and camk2g1, are necessary for convergent extension (CE) cell movements. CaMKII morphant phenotypes were observed as early as epiboly. At the 1-3 somite stage, neuroectoderm and paraxial cells remained unconverged in both morphants. Later, somites lacked their stereotypical shape and were wider, more closely spaced, and body gap angles increased. At 24hpf, somite compression and notochord undulation coincided with a shorter and broader body axis. A camk2b1 crispant was generated which phenocopied the camk2b1 morphant. The levels of cell proliferation, apoptosis and paraxial and neuroectodermal markers were unchanged in morphants. Hyperactivation of CaMKII during gastrulation by transient pharmacological intervention (thapsigargin) also caused CE defects. Mosaically expressed dominant-negative CaMKII recapitulated these phenotypes and showed significant midline bifurcation. Finally, the introduction of CaMKII partially rescued Wnt11 morphant phenotypes. CONCLUSIONS: Overall, these data support a model whereby cyclically activated CaMKII encoded from two genes enables cell migration during the process of CE.

RNA sequencing of isolated cell populations expressing human APOL1 G2 risk variant reveals molecular correlates of sickle cell nephropathy in zebrafish podocytes.

Kidney failure occurs in 5-13% of individuals with sickle cell disease and is associated with early mortality. Two APOL1 alleles (G1 and G2) have been identified as risk factors for sickle cell disease nephropathy. Both risk alleles are prevalent in individuals with recent African ancestry and have been associated with nephropathic complications in other diseases. Despite the association of G1 and G2 with kidney dysfunction, the mechanisms by which these variants contribute to increased risk remain poorly understood. Previous work in zebrafish models suggest that the G2 risk allele functions as a dominant negative, whereas the G1 allele is a functional null. To understand better the cellular pathology attributed to APOL1 G2, we investigated the in vivo effects of the G2 risk variant on distinct cell types using RNA sequencing. We surveyed APOL1 G2 associated transcriptomic alterations in podocytes and vascular endothelial cells isolated from zebrafish larvae expressing cell-type specific reporters. Our analysis identified many transcripts (n = 7,523) showing differential expression between APOL1 G0 (human wild-type) and APOL1 G2 exposed podocytes. Conversely, relatively few transcripts (n = 107) were differentially expressed when comparing APOL1 G0 and APOL1 G2 exposed endothelial cells. Pathway analysis of differentially expressed transcripts in podocytes showed enrichment for autophagy associated terms such as "Lysosome" and "Phagosome", implicating these pathways in APOL1 G2 associated kidney dysfunction. This work provides insight into the molecular pathology of APOL1 G2 nephropathy which may offer new therapeutic strategies for multiple disease contexts such as sickle cell nephropathy.

Loss-of-Function Mutations in UNC45A Cause a Syndrome Associating Cholestasis, Diarrhea, Impaired Hearing, and Bone Fragility.

Despite the rapid discovery of genes for rare genetic disorders, we continue to encounter individuals presenting with syndromic manifestations. Here, we have studied four affected people in three families presenting with cholestasis, congenital diarrhea, impaired hearing, and bone fragility. Whole-exome sequencing of all affected individuals and their parents identified biallelic mutations in Unc-45 Myosin Chaperone A (UNC45A) as a likely driver for this disorder. Subsequent in vitro and in vivo functional studies of the candidate gene indicated a loss-of-function paradigm, wherein mutations attenuated or abolished protein activity with concomitant defects in gut development and function.

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos.

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr. Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol. This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer's Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Newborn screening and the era of medical genomics.

Across the span of the last 75+ years, technological and conceptual advances in genetics have found rapid implementation at the beginning of human life. From karyotype testing, to molecular cytogenetics, to gene panel testing, and now to whole exome and whole genome sequencing, each iterative expansion of our capability to acquire genetic data on the next generation has been implemented quickly in the clinical setting. In tandem, our continuously expanding ability to acquire large volumes of genetic data has generated its own challenges in terms of interpretation, clinical utility of the information, and concerns over privacy and discrimination; for the first time, we are faced with the possibility of having complete access to our genetic data from birth, if not shortly after conception. Here, we discuss the evolution of the field toward this new reality and we consider the potentially far-reaching consequences and, at present, an unclear path toward developing best practices for implementation.

A human laterality disorder caused by a homozygous deleterious mutation in MMP21.

BACKGROUND: Laterality in the vertebrate embryo is determined by left-right asymmetric gene expression driven by the flow of extraembryonic fluid across the embryonic node. Defects in these processes cause heterotaxy, the abnormal formation and arrangement of visceral organs that can range from complete inversion of symmetry to the selective misarrangement of organs. However, our understanding of the genetic causality for laterality defects in human beings remains relatively limited. METHODS: We performed whole exome sequencing in a consanguineous family with heterotaxia. To interrogate the pathogenic potential of the discovered variant, we used an in vivo system in which the potential of the candidate gene to induce L-R asymmetry was tested by transient suppression and CRISPR/Cas9-induced deletions. We also used in vitro assays to test a possible link between our exome-derived candidate and Notch signaling. RESULTS: We identified a homozygous 2 bp deletion in MMP21, encoding matrix metalloproteinase-21, as the sole coding mutation that segregated with the phenotype. Transient suppression or CRISPR/Cas9-mediated deletion of mmp21 in zebrafish embryos induced cardiac looping defects, with concomitant disruption of laterality markers in the lateral plate mesoderm and disrupted notch signalling in vitro and in vivo. CONCLUSIONS: Our data implicate loss of MMP21 as a cause of heterotaxy in humans with concomitant defects in Notch signaling. In support of this finding, a homozygous missense mutation in MMP21 was identified previously in mice with N-Ethyl-N-Nitrosourea (ENU)-induced heterotaxy. Taken together, these observations suggest a role of matrix metalloproteinases in the establishment of asymmetric organ development, likely through the regulation of morphogenetic signals.

Exome Sequence Analysis Suggests that Genetic Burden Contributes to Phenotypic Variability and Complex Neuropathy.

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous distal symmetric polyneuropathy. Whole-exome sequencing (WES) of 40 individuals from 37 unrelated families with CMT-like peripheral neuropathy refractory to molecular diagnosis identified apparent causal mutations in ∼ 45% (17/37) of families. Three candidate disease genes are proposed, supported by a combination of genetic and in vivo studies. Aggregate analysis of mutation data revealed a significantly increased number of rare variants across 58 neuropathy-associated genes in subjects versus controls, confirmed in a second ethnically discrete neuropathy cohort, suggesting that mutation burden potentially contributes to phenotypic variability. Neuropathy genes shown to have highly penetrant Mendelizing variants (HPMVs) and implicated by burden in families were shown to interact genetically in a zebrafish assay exacerbating the phenotype established by the suppression of single genes. Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity.

Missense mutations in TENM4, a regulator of axon guidance and central myelination, cause essential tremor.

Essential tremor (ET) is a common movement disorder with an estimated prevalence of 5% of the population aged over 65 years. In spite of intensive efforts, the genetic architecture of ET remains unknown. We used a combination of whole-exome sequencing and targeted resequencing in three ET families. In vitro and in vivo experiments in oligodendrocyte precursor cells and zebrafish were performed to test our findings. Whole-exome sequencing revealed a missense mutation in TENM4 segregating in an autosomal-dominant fashion in an ET family. Subsequent targeted resequencing of TENM4 led to the discovery of two novel missense mutations. Not only did these two mutations segregate with ET in two additional families, but we also observed significant over transmission of pathogenic TENM4 alleles across the three families. Consistent with a dominant mode of inheritance, in vitro analysis in oligodendrocyte precursor cells showed that mutant proteins mislocalize. Finally, expression of human mRNA harboring any of three patient mutations in zebrafish embryos induced defects in axon guidance, confirming a dominant-negative mode of action for these mutations. Our genetic and functional data, which is corroborated by the existence of a Tenm4 knockout mouse displaying an ET phenotype, implicates TENM4 in ET. Together with previous studies of TENM4 in model organisms, our studies intimate that processes regulating myelination in the central nervous system and axon guidance might be significant contributors to the genetic burden of this disorder.

Recessive mutations in the α3 (VI) collagen gene COL6A3 cause early-onset isolated dystonia.

Isolated dystonia is a disorder characterized by involuntary twisting postures arising from sustained muscle contractions. Although autosomal-dominant mutations in TOR1A, THAP1, and GNAL have been found in some cases, the molecular mechanisms underlying isolated dystonia are largely unknown. In addition, although emphasis has been placed on dominant isolated dystonia, the disorder is also transmitted as a recessive trait, for which no mutations have been defined. Using whole-exome sequencing in a recessive isolated dystonia-affected kindred, we identified disease-segregating compound heterozygous mutations in COL6A3, a collagen VI gene associated previously with muscular dystrophy. Genetic screening of a further 367 isolated dystonia subjects revealed two additional recessive pedigrees harboring compound heterozygous mutations in COL6A3. Strikingly, all affected individuals had at least one pathogenic allele in exon 41, including an exon-skipping mutation that induced an in-frame deletion. We tested the hypothesis that disruption of this exon is pathognomonic for isolated dystonia by inducing a series of in-frame deletions in zebrafish embryos. Consistent with our human genetics data, suppression of the exon 41 ortholog caused deficits in axonal outgrowth, whereas suppression of other exons phenocopied collagen deposition mutants. All recessive mutation carriers demonstrated early-onset segmental isolated dystonia without muscular disease. Finally, we show that Col6a3 is expressed in neurons, with relevant mRNA levels detectable throughout the adult mouse brain. Taken together, our data indicate that loss-of-function mutations affecting a specific region of COL6A3 cause recessive isolated dystonia with underlying neurodevelopmental deficits and highlight the brain extracellular matrix as a contributor to dystonia pathogenesis.

Mutations in RAD21 disrupt regulation of APOB in patients with chronic intestinal pseudo-obstruction.

BACKGROUND & AIMS: Chronic intestinal pseudo-obstruction (CIPO) is characterized by severe intestinal dysmotility that mimics a mechanical subocclusion with no evidence of gut obstruction. We searched for genetic variants associated with CIPO to increase our understanding of its pathogenesis and to identify potential biomarkers. METHODS: We performed whole-exome sequencing of genomic DNA from patients with familial CIPO syndrome. Blood and lymphoblastoid cells were collected from patients and controls (individuals without CIPO); levels of messenger RNA (mRNA) and proteins were analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblot, and mobility shift assays. Complementary DNAs were transfected into HEK293 cells. Expression of rad21 was suppressed in zebrafish embryos using a splice-blocking morpholino (rad21a). Gut tissues were collected and analyzed. RESULTS: We identified a homozygous mutation (p.622, encodes Ala>Thr) in RAD21 in patients from a consanguineous family with CIPO. Expression of RUNX1, a target of RAD21, was reduced in cells from patients with CIPO compared with controls. In zebrafish, suppression of rad21a reduced expression of runx1; this phenotype was corrected by injection of human RAD21 mRNA, but not with the mRNA from the mutated p.622 allele. rad21a Morpholino zebrafish had delayed intestinal transit and greatly reduced numbers of enteric neurons, similar to patients with CIPO. This defect was greater in zebrafish with suppressed expression of ret and rad21, indicating their interaction in the regulation of gut neurogenesis. The promoter region of APOB bound RAD21 but not RAD21 p.622 Ala>Thr; expression of wild-type RAD21 in HEK293 cells repressed expression of APOB, compared with control vector. The gut-specific isoform of APOB (APOB48) is overexpressed in sera from patients with CIPO who carry the RAD21 mutation. APOB48 also is overexpressed in sporadic CIPO in sera and gut biopsy specimens. CONCLUSIONS: Some patients with CIPO carry mutations in RAD21 that disrupt the ability of its product to regulate genes such as RUNX1 and APOB. Reduced expression of rad21 in zebrafish, and dysregulation of these target genes, disrupts intestinal transit and the development of enteric neurons.

Functionally compromised CHD7 alleles in patients with isolated GnRH deficiency.

Inactivating mutations in chromodomain helicase DNA binding protein 7 (CHD7) cause CHARGE syndrome, a severe multiorgan system disorder of which Isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD) is a minor feature. Recent reports have described predominantly missense CHD7 alleles in IGD patients, but it is unclear if these alleles are relevant to causality or overall genetic burden of Kallmann syndrome (KS) and normosmic form of IGD. To address this question, we sequenced CHD7 in 783 well-phenotyped IGD patients lacking full CHARGE features; we identified nonsynonymous rare sequence variants in 5.2% of the IGD cohort (73% missense and 27% splice variants). Functional analyses in zebrafish using a surrogate otolith assay of a representative set of these CHD7 alleles showed that rare sequence variants observed in controls showed no altered function. In contrast, 75% of the IGD-associated alleles were deleterious and resulted in both KS and normosmic IGD. In two families, pathogenic mutations in CHD7 coexisted with mutations in other known IGD genes. Taken together, our data suggest that rare deleterious CHD7 alleles contribute to the mutational burden of patients with both KS and normosmic forms of IGD in the absence of full CHARGE syndrome. These findings (i) implicate a unique role or preferential sensitivity for CHD7 in the ontogeny of GnRH neurons, (ii) reiterate the emerging genetic complexity of this family of IGD disorders, and (iii) demonstrate how the coordinated use of well-phenotyped cohorts, families, and functional studies can inform genetic architecture and provide insights into the developmental biology of cellular systems.

Disruptive CHD8 mutations define a subtype of autism early in development.

Autism spectrum disorder (ASD) is a heterogeneous disease in which efforts to define subtypes behaviorally have met with limited success. Hypothesizing that genetically based subtype identification may prove more productive, we resequenced the ASD-associated gene CHD8 in 3,730 children with developmental delay or ASD. We identified a total of 15 independent mutations; no truncating events were identified in 8,792 controls, including 2,289 unaffected siblings. In addition to a high likelihood of an ASD diagnosis among patients bearing CHD8 mutations, characteristics enriched in this group included macrocephaly, distinct faces, and gastrointestinal complaints. chd8 disruption in zebrafish recapitulates features of the human phenotype, including increased head size as a result of expansion of the forebrain/midbrain and impairment of gastrointestinal motility due to a reduction in postmitotic enteric neurons. Our findings indicate that CHD8 disruptions define a distinct ASD subtype and reveal unexpected comorbidities between brain development and enteric innervation.

CaMK-II activation is essential for zebrafish inner ear development and acts through Delta-Notch signaling.

Zebrafish inner ear development is characterized by the crystallization of otoliths onto immotile kinocilia that protrude from sensory "hair" cells. The stereotypical formation of these sensory structures is dependent on the expression of key patterning genes and on Ca2+ signals. One potential target of Ca2+ signaling in the inner ear is the type II Ca2+/calmodulin-dependent protein kinase (CaMK-II), which is preferentially activated in hair cells, with intense activation at the base of kinocilia. In zebrafish, CaMK-II is encoded by seven genes; the expression of one of these genes (camk2g1) is enriched in hair cells. The suppression of camk2g1 expression by antisense morpholino oligonucleotides or inhibition of CaMK-II activation by the pharmacological antagonist, KN-93, results in aberrant otolith formation without preventing cilia formation. In fact, CaMK-II suppression results in additional ciliated hair cells and altered levels of Delta-Notch signaling members. DeltaA and deltaD transcripts are increased and DeltaD protein accumulates in hair cells of CaMK-II morphants, indicative of defective recycling and/or exocytosis. Our findings indicate that CaMK-II plays a critical role in the developing ear, influencing cell differentiation through extranuclear effects on Delta-Notch signaling. Continued expression and activation of CaMK-II in maculae and cristae in older embryos suggests continued roles in auditory sensory maturation and transduction.

CaMK-II is a PKD2 target that promotes pronephric kidney development and stabilizes cilia.

Intracellular Ca²âº signals influence gastrulation, neurogenesis and organogenesis through pathways that are still being defined. One potential Ca²âº mediator of many of these morphogenic processes is CaMK-II, a conserved calmodulin-dependent protein kinase. Prolonged Ca²âº stimulation converts CaMK-II into an activated state that, in the zebrafish, is detected in the forebrain, ear and kidney. Autosomal dominant polycystic kidney disease has been linked to mutations in the Ca²âº-conducting TRP family member PKD2, the suppression of which in vertebrate model organisms results in kidney cysts. Both PKD2-deficient and CaMK-II-deficient zebrafish embryos fail to form pronephric ducts properly, and exhibit anterior cysts and destabilized cloacal cilia. PKD2 suppression inactivates CaMK-II in pronephric cells and cilia, whereas constitutively active CaMK-II restores pronephric duct formation in pkd2 morphants. PKD2 and CaMK-II deficiencies are synergistic, supporting their existence in the same genetic pathway. We conclude that CaMK-II is a crucial effector of PKD2 Ca²âº that both promotes morphogenesis of the pronephric kidney and stabilizes primary cloacal cilia.

The activation of membrane targeted CaMK-II in the zebrafish Kupffer's vesicle is required for left-right asymmetry.

Intracellular calcium ion (Ca(2+)) elevation on the left side of the mouse embryonic node or zebrafish Kupffer's vesicle (KV) is the earliest asymmetric molecular event that is functionally linked to lateral organ placement in these species. In this study, Ca(2+)/CaM-dependent protein kinase (CaMK-II) is identified as a necessary target of this Ca(2+) elevation in zebrafish embryos. CaMK-II is transiently activated in approximately four interconnected cells along the anterior left wall of the KV between the six- and 12-somite stages, which is coincident with known left-sided Ca(2+) elevations. Within these cells, activated CaMK-II is observed at the surface and in clusters, which appear at the base of some KV cilia. Although seven genes encode catalytically active CaMK-II in early zebrafish embryos, one of these genes also encodes a truncated inactive variant (alphaKAP) that can hetero-oligomerize with and target active enzyme to membranes. alphaKAP, beta2 CaMK-II and gamma1 CaMK-II antisense morpholino oligonucleotides, as well as KV-targeted dominant negative CaMK-II, randomize organ laterality and southpaw (spaw) expression in lateral plate mesoderm (LPM). Left-sided CaMK-II activation was most dependent on an intact KV, the PKD2 Ca(2+) channel and gamma1 CaMK-II; however, alphaKAP, beta2 CaMK-II and the RyR3 ryanodine receptor were also necessary for full CaMK-II activation. This is the first report to identify a direct Ca(2+)-sensitive target in left-right asymmetry and supports a model in which membrane targeted CaMK-II hetero-oligomers in nodal cells transduce the left-sided PKD2-dependent Ca(2+) signals to the LPM.

Tbx5-mediated expression of Ca(2+)/calmodulin-dependent protein kinase II is necessary for zebrafish cardiac and pectoral fin morphogenesis.

Mutations in the T-box transcription factor, TBX5, result in Holt-Oram syndrome (HOS), a human condition in which cardiac development is defective and forelimbs are stunted. Similarly, zebrafish tbx5 morphants and mutants (heartstrings; hst) lack pectoral fins and exhibit a persistently elongated heart that does not undergo chamber looping. Tbx5 is expressed in the developing atrium, ventricle and in pectoral fin fields, but its genetic targets are still being uncovered. In this study, evidence is provided that Tbx5 induces the expression of a specific member of the CaMK-II (the type II multifunctional Ca(2+)/calmodulin-dependent protein kinase) family; this CaMK-II is necessary for proper heart and fin development. Morphants of beta2 CaMK-II (camk2b2), but not the beta1 CaMK-II (camk2b1) paralog, exhibit bradycardia, elongated hearts and diminished pectoral fin development. Normal cardiac phenotypes can be restored by ectopic cytosolic CaMK-II expression in tbx5 morphants. Like tbx5, camk2b2 is expressed in the pectoral fin and looping heart, but this expression is diminished in both tbx5 morphant and hst embryos. Conversely, the introduction of excess Tbx5 into zebrafish embryos and mouse fibroblasts doubles CaMK-II expression. We conclude that beta CaMK-II expression and activity are necessary for proper cardiac and limb morphogenesis. These findings not only identify a morphogenic target for Ca(2+) during heart development, but support implied roles for CaMK-II in adult heart remodeling.