Microcephaly-dystonia due to mutated PLEKHG2 with impaired actin polymerization.

Rearrangement of the actin cytoskeleton is controlled by RhoGTPases which are activated by RhoGEFs. We identified homozygosity for Arg204Trp mutation in the Rho guanidine exchange factor (RhoGEF) PLEKHG2 gene in five patients with profound mental retardation, dystonia, postnatal microcephaly, and distinct neuroimaging pattern. The activity of the mutant PLEKHG2 was significantly decreased, both in basal state and when Gßγ- or lysophosphatidic acid (LPA)-stimulated. SDF1a-stimulated actin polymerization was significantly impaired in patient cells, and this abnormality was duplicated in control cells when PLEKHG2 expression was downregulated. These results underscore the role of PLEKHG2 in actin polymerization and delineate the clinical and radiological findings in PLEKHG2 deficiency.

Cytokine secretion and NK cell activity in human ADAM17 deficiency.

Genetic deficiencies provide insights into gene function in humans. Here we describe a patient with a very rare genetic deficiency of ADAM17. We show that the patient's PBMCs had impaired cytokine secretion in response to LPS stimulation, correlating with the clinical picture of severe bacteremia from which the patient suffered. ADAM17 was shown to cleave CD16, a major NK killer receptor. Functional analysis of patient's NK cells demonstrated that his NK cells express normal levels of activating receptors and maintain high surface levels of CD16 following mAb stimulation. Activation of individual NK cell receptors showed that the patient's NK cells are more potent when activated directly by CD16, albeit no difference was observed in Antibody Depedent Cytotoxicity (ADCC) assays. Our data suggest that ADAM17 inhibitors currently considered for clinical use to boost CD16 activity should be cautiously applied, as they might have severe side effects resulting from impaired cytokine secretion.

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.

Mutations in SLC1A4, encoding the brain serine transporter, are associated with developmental delay, microcephaly and hypomyelination.

BACKGROUND: L-serine plays an essential role in neuronal development and function. Although a non-essential amino acid, L-serine must be synthesised within the brain because of its poor permeability by the blood-brain barrier. Within the brain, its synthesis is confined to astrocytes, and its shuttle to neuronal cells is performed by a dedicated neutral amino acid transporter, ASCT1. METHODS AND RESULTS: Using exome analysis we identified the recessive mutations, p.E256K, p.L315fs, and p.R457W, in SLC1A4, the gene encoding ASCT1, in patients with developmental delay, microcephaly and hypomyelination; seizure disorder was variably present. When expressed in a heterologous system, the mutations did not affect the protein level at the plasma membrane but abolished or markedly reduced L-serine transport for p.R457W and p.E256K mutations, respectively. Interestingly, p.E256K mutation displayed a lower L-serine and alanine affinity but the same substrate selectivity as wild-type ASCT1. CONCLUSIONS: The clinical phenotype of ASCT1 deficiency is reminiscent of defects in L-serine biosynthesis. The data underscore that ASCT1 is essential in brain serine transport. The SLC1A4 p.E256K mutation has a carrier frequency of 0.7% in the Ashkenazi-Jewish population and should be added to the carrier screening panel in this community.

Deleterious mutation in SYCE1 is associated with non-obstructive azoospermia.

PURPOSE: To determine the molecular basis of familial, autosomal-recessive, non-obstructive azoospermia in a consanguineous Iranian Jewish family. METHODS: We investigated the genetic cause of non-obstructive azoospermia in two affected siblings from a consanguineous family. Homozygosity mapping in the DNA samples of the patients and their normospermic brother was followed by exome analysis of one of the patients. Other family members were genotyped for the mutation by Sanger sequencing. The mutation effect was demonstrated by immunostaining of the patients' testicular tissue. RESULTS: The two patients were homozygous for a splice site mutation in SYCE1 which resulted in retention of intron three in the cDNA and premature stop codon. SYCE1 encodes a Synaptonemal Complex protein which plays an essential role during meiosis. Immunostaining of patient's testicular tissue with anti-Syce1 antibody revealed an undetectable level of Syce1. Histological examination of the patients' tissue disclosed immature-stages spermatocytes without mature forms, indicating maturation arrest. CONCLUSION: The significance of most synaptonemal complex proteins was previously demonstrated in a mutant mouse model. The present report underscores the importance of synaptonemal complex proteins in spermatogenenesis in humans. Our new approach, combining homozygosity mapping and exome sequencing, resulted in one of the first reports of an autosomal-recessive form of NOA.

A defect in the retromer accessory protein, SNX27, manifests by infantile myoclonic epilepsy and neurodegeneration.

The composition of the neuronal cell surface dictates synaptic plasticity and thereby cognitive development. This remodeling of the synapses is governed by the endocytic network which internalize transmembrane proteins, then sort them back to the cell surface or carry them to the lysosome for degradation. The multi-protein retromer complex is central to this selection, capturing specific transmembrane proteins and remodeling the cell membrane to form isolated cargo-enriched transport carriers. We investigated a consanguineous family with four patients who presented in infancy with intractable myoclonic epilepsy and lack of psychomotor development. Using exome analysis, we identified a homozygous deleterious mutation in SNX27, which encodes sorting nexin 27, a retromer cargo adaptor. In western analysis of patient fibroblasts, the encoded mutant protein was expressed at an undetectable level when compared with a control sample. The patients' presentation and clinical course recapitulate that reported for the SNX27 knock-out mouse. Since the cargo proteins for SNX27-mediated sorting include subunits of ionotropic glutamate receptors and endosome-to-cell surface synaptic insertion of AMPA receptors is severely perturbed in SNX27(-/-) neurons, it is proposed that at least part of the neurological aberrations observed in the patients is attributed to defective sorting of ionotropic glutamate receptors. SNX27 deficiency is now added to the growing list of neurodegenerative disorders associated with retromer dysfunction.

A human laterality disorder associated with a homozygous WDR16 deletion.

The laterality in the embryo is determined by left-right asymmetric gene expression driven by the flow of extraembryonic fluid, which is maintained by the rotary movement of monocilia on the nodal cells. Defects manifest by abnormal formation and arrangement of visceral organs. The genetic etiology of defects not associated with primary ciliary dyskinesia is largely unknown. In this study, we investigated the cause of situs anomalies, including heterotaxy syndrome and situs inversus totalis, in a consanguineous family. Whole-exome analysis revealed a homozygous deleterious deletion in the WDR16 gene, which segregated with the phenotype. WDR16 protein was previously proposed to play a role in cilia-related signal transduction processes; the rat Wdr16 protein was shown to be confined to cilia-possessing tissues and severe hydrocephalus was observed in the wdr16 gene knockdown zebrafish. The phenotype associated with the homozygous deletion in our patients suggests a role for WDR16 in human laterality patterning. Exome analysis is a valuable tool for molecular investigation even in cases of large deletions.

Hindbrain malformation and myoclonic seizures associated with a deleterious mutation in the INPP4A gene.

Hindbrain malformations with predominant cerebellar involvement have many causes including chromosomal disorders, specific genetic syndromes, and prenatal disruptions. The combination of a hindbrain malformation and myoclonic epilepsy is rare. Using exome sequencing in a consanguineous family, we identified a homozygous genomic deletion of 1770 bp within the INPP4A gene in a patient with myoclonic epilepsy, microcephaly, and atrophy of the inferior vermis and cerebellum. INPP4A participates in the excitatory glutamate signaling pathway and is essential for the degradation of phosphatidylinositol (3,4)-bisphosphate. Glutamatergic signaling is important for hindbrain development and is implicated in the pathogenesis of epilepsy, as well as excitotoxic cell death. Indeed, excessive glutamatergic stimulation was previously reported in INPP4A knockout mice. Our data adds a new etiology to the spectrum of hindbrain malformations in human, and when presented with myoclonic epilepsy may lead to the clinical suspicion of INPP4A defect. The present report further underscores the importance of phosphoinositides for the development of the inferior cerebellum and vermis.

Truncating mutation in the nitric oxide synthase 1 gene is associated with infantile achalasia.

Nitric oxide is thought to have a role in the pathogenesis of achalasia. We performed a genetic analysis of 2 siblings with infant-onset achalasia. Exome analysis revealed that they were homozygous for a premature stop codon in the gene encoding nitric oxide synthase 1. Kinetic analyses and molecular modeling showed that the truncated protein product has defects in folding, nitric oxide production, and binding of cofactors. Heller myotomy had no effect in these patients, but sildenafil therapy increased their ability to drink. The finding recapitulates the previously reported phenotype of nitric oxide synthase 1-deficient mice, which have achalasia. Nitric oxide signaling appears to be involved in the pathogenesis of achalasia in humans.

TRMT10A dysfunction is associated with abnormalities in glucose homeostasis, short stature and microcephaly.

BACKGROUND: Trm10 is a tRNA m(1)G9 methyltransferase, which in yeast modifies 12 different tRNA species, yet is considered non-essential for viability under standard growth conditions. In humans, there are three Trm10 orthologs, one mitochondrial and two presumed cytoplasmic. A nonsense mutation in one of the cytoplasmic orthologs (TRMT10A) has recently been associated with microcephaly, intellectual disability, short stature and adolescent onset diabetes. METHODS AND RESULTS: The subjects were three patients who suffered from microcephaly, intellectual disability, short stature, delayed puberty, seizures and disturbed glucose metabolism, mainly hyperinsulinaemic hypoglycaemia. A homozygous Gly206Arg (G206R) mutation in the TRMT10A gene was identified using whole exome sequencing. The mutation segregated in the family and was absent from large control cohorts. Determination of the methylation activity of the expressed wild-type (WT) and variant TRMT10A enzymes with transcripts of (32)P -tRNA(Gly) GCC as a substrate revealed a striking defect (<0.1% of WT activity) for the variant enzyme. The binding affinity of the G206R variant enzyme to tRNA, determined by fluorescence anisotropy, was similar to that of the WT enzyme. CONCLUSIONS: The completely abolished m(1)G9 methyltransferase activity of the mutant enzyme is likely due to significant defects in its ability to bind the methyl donor S-adenosyl methionine. We propose that TRMT10A deficiency accounts for abnormalities in glucose homeostasis initially manifesting both ketotic and non-ketotic hypoglycaemic events with transition to diabetes in adolescence, perhaps as a consequence of accelerated ß cell apoptosis. The seizure disorder and intellectual disability are probably secondary to mutant gene expression in neuronal tissue.

Extracellular matrix constituents interfere with Newcastle disease virus spread in solid tissue and diminish its potential oncolytic activity.

Advanced melanoma cells, characterized by resistance to chemotherapy, have been shown to be highly sensitive to oncolysis by Newcastle disease virus (NDV). In the present study, we investigated the capacity of NDV to specifically infect and spread into solid tissues of human melanoma and lung carcinoma, in vivo and ex vivo. For this purpose a new model of SCID-beige mice implanted with human melanoma was developed. Surprisingly, the replication competent NDV-MTH and the attenuated, single-cycle replication NDV-HUJ strains, demonstrated a similar oncolytic activity in the melanoma-implanted mice. Further, ex vivo analysis, using organ cultures derived from the melanoma tissues indicated a limited spread of the two NDV strains in the tissue. Extracellular matrix (ECM) molecules, notably heparin sulfate and collagen, were found to limit viral spread in the tissue. This observation was validated with yet another solid tumour of human lung carcinoma. Taken together, the results indicate that the ECM acts as a barrier to virus spread within solid tumour tissues and that this restriction must be overcome to achieve effective oncolysis with NDV.

Characterization of factors that determine lentiviral vector tropism in skin tissue using an ex vivo model.

BACKGROUND: Lentiviral tropism to a solid tissue may be determined by receptor availability, the differentiation state of cells and the three-dimensional architecture of the tissue. METHODS: Using skin organ cultures, lentiviral vector tropism was compared with that of keratinocytes in cell culture. Furthermore, the tropism of lentiviral vector to mouse and human tissues was compared ex vivo, in attempt to validate the mouse skin as an experimental system for human gene therapy of skin diseases. RESULTS: The results obtained indicated that although early progenitor keratinocytes (keratin 15+ and p63+), when grown in culture are permissive to lentiviral vector, they are resistant to transduction in their native 'niche' in the skin tissue. Transiently amplifying keratinocytes (keratin 14+) on the other hand, are permissive to lentiviral vector transduction, in cell culture and in the skin, after separation of the epidermis from the dermis layer. Keratinocytes (keratin 14+) in the hair follicle of human skin are resistant to lentiviral transduction, even after partial digestion of the extracellular matrix collagen. By contrast, collagenase pretreatment of mouse tissue facilitated transduction of keratinocytes within the hair follicle. Because lentivirus pseudotyped by two envelopes (amphotropic murine leukemia virus and vesicular stomatitis virus G glycoprotein) display the same tropism, we suggest that receptor availability is not the critical factor in the pattern of skin tissue transduction. CONCLUSIONS: Taken together, the results obtained in the present study indicate that lentiviral vector tropism in the three-dimensional skin tissue is distinct from the tropism to keratinocytes in culture and is dependent on a complex interplay of extracellular restrictions.

The oncolytic activity of Newcastle disease virus NDV-HUJ on chemoresistant primary melanoma cells is dependent on the proapoptotic activity of the inhibitor of apoptosis protein Livin.

Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. In contrast, melanoma tumor cells that do not express Livin are relatively resistant to NDV-HUJ treatment. Furthermore, we show that NDV-HUJ-induced oncolysis is attributed to the dual function of Livin: although Livin inhibits apoptosis through the inhibition of caspases, under the robust apoptotic stimulation of NDV-HUJ, caspases can cleave Livin to create a truncated protein with a paradoxical proapoptotic activity. Thus, NDV-HUJ is a potent inducer of apoptosis that can overcome the antiapoptotic effect of Livin and allow cleavage of Livin into the proapoptotic tLivin protein. Moreover, the results indicate that the interferon system, which is functional in melanoma, is not involved in NDV-induced oncolysis. Taken together, our data offer the possibility of a new viral oncolytic treatment for chemoresistant melanoma.

Tropism of lentiviral vectors in skin tissue.

The skin is an attractive tissue for gene therapy applications to treat genetic disorders and to express systemically delivered transgenes encoding therapeutic proteins. Understanding the tissue tropism of vectors is a prerequisite for the design of gene therapy trials. Using an ex vivo system of organ culture, we studied factors that determined viral tropism to the epidermal and dermal cells in human and mouse skin. We applied in these studies a lentiviral vector pseudotyped with two glycoproteins that use different cell receptors (vesicular stomatitis virus glycoprotein [VSV-G] and amphotropic murine leukemia virus envelope). The extent of infection with the amphotropic pseudotype was much higher than that of VSV-G, especially at low multiplicities of infection. In contrast, the tropism of these two pseudotypes in skin tissues was similar; at low multiplicities the infection was limited to areas near the basal layer of the epidermis, whereas at high multiplicities the infection extended to the dermal layer. To overcome physical barriers in the skin, the epidermal and dermal layers were separated and infected. Whereas the human epidermis was readily infected, we could not detect infection of stem and early progenitor cells in their niche. In contrast, mouse epidermis was completely resistant to infection. Dermal cells of both species were readily infected with the two pseudotypes. Molecular analysis indicated that infection of mouse epidermal cells was restricted after proviral DNA synthesis and before integration. In conclusion, we show that lentiviral tropism in a solid tissue is dependent on several factors, extra- and intracellular, distinct of the cellular receptors.