Please cite this article as: M. Lukacs, J. Gilley, Y. Zhu, et al., Severe biallelic lossof-function mutations in nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) in two fetuses with fetal akinesia deformation sequence, Experimental Neurology,
The glycosylphosphatidylinositol (GPI) anchor is a post-translational modification added to approximately 150 different proteins to facilitate proper membrane anchoring and trafficking to lipid rafts. Biosynthesis and remodeling of the GPI anchor requires the activity of over 20 distinct genes. Defects in the biosynthesis of GPI anchors in humans lead to inherited glycosylphosphatidylinositol deficiency (IGD). IGD patients display a wide range of phenotypes though the central nervous system (CNS) appears to be the most commonly affected tissue. A full understanding of the etiology of these phenotypes has been hampered by the lack of animal models due to embryonic lethality of GPI biosynthesis gene null mutants. Here we model IGD by genetically ablating GPI production in the CNS with a conditional mouse allele of phosphatidylinositol glycan anchor biosynthesis, class A (Piga) and Nestin-Cre. We find that the mutants do not have structural brain defects but do not survive past weaning. The mutants show progressive decline with severe ataxia consistent with defects in cerebellar development. We show that the mutants have reduced myelination and defective Purkinje cell development. Surprisingly, we found that Piga was expressed in a fairly restricted pattern in the early postnatal brain consistent with the defects we observed in our model. Thus, we have generated a novel mouse model of the neurological defects of IGD which demonstrates a critical role for GPI biosynthesis in cerebellar and white matter development.
Glycosylphosphatidylinositol (GPI) anchors attach nearly 150 proteins to the cell membrane. Patients with pathogenic variants in GPI biosynthesis genes develop diverse phenotypes including seizures, dysmorphic facial features and cleft palate through an unknown mechanism. We identified a novel mouse mutant (cleft lip/palate, edema and exencephaly; Clpex) with a hypo-morphic mutation in Post-Glycophosphatidylinositol Attachment to Proteins-2 (Pgap2), a component of the GPI biosynthesis pathway. The Clpex mutation decreases surface GPI expression. Surprisingly, Pgap2 showed tissue-specific expression with enrichment in the brain and face. We found the Clpex phenotype is due to apoptosis of neural crest cells (NCCs) and the cranial neuroepithelium. We showed folinic acid supplementation in utero can partially rescue the cleft lip phenotype. Finally, we generated a novel mouse model of NCC-specific total GPI deficiency. These mutants developed median cleft lip and palate demonstrating a previously undocumented cell autonomous role for GPI biosynthesis in NCC development.
Flax undergoes heritable genomic changes in response to nutrient stress, including changes in total DNA content, rDNA copy number variation, and the appearance of Linum Insertion Sequence 1 (LIS-1). The nature of the genomic changes suggests a very different mechanism, which is not yet understood, from that of other DNA changes in response to stress, such as the activation of transposable elements. To identify the genes that control genomic changes in response to stress in flax, reciprocal crosses were made between a responsive flax line, Stormont cirrus, and an unresponsive line, Bethune. The ability of the F2 generation (from selfed F1 plants) to respond to nutrient stress was assayed using the insertion of LIS-1 as the criteria for responsiveness. Twenty-nine out of 89 F2s responded at 5 weeks, suggesting that 3-4 dominant loci were all necessary for early LIS-1 insertion. Seventy out of 76 responded at 10 weeks, indicating two dominant loci independently capable of initiating LIS-1 insertion under prolonged nutrient stress. F1 plants and their progeny with either Pl or Bethune as the maternal parent were capable of responding with LIS-1 insertion, indicating that LIS-1 insertion is under nuclear genetic control and does not involve maternal factors. Thus, a small number of loci within the genome of Stormont cirrus appear to control the ability to respond to nutrient stress with LIS-1 insertion. A genetic map of the flax genome is currently under construction, and will be used to identify these loci within the genome.
Whole exome sequencing continues to end the diagnostic odyssey for a number of patients and expands our knowledge of phenotypes associated with gene mutations. We describe an 11-year-old female patient with a constellation of symptoms including congenital cataracts, gut dysmotility, sensory neuropathy, and bifrontal polymicrogyria. Whole exome sequencing was performed and identified a de novo heterozygous missense mutation in the ATPase motor domain of cytoplasmic dynein heavy chain 1 (DYNC1H1), which is known to be involved in neuronal migration and retrograde axonal transport. The mutation was found to be highly damaging by multiple prediction programs. The residue is highly conserved, and reported mutations in this gene result in a variety of phenotypes similar to that of our patient. We report only the second case of congenital cataracts and the first of gut dysmotility in a patient with DYNC1H1, thus expanding the spectrum of disease seen in DYNC1H1 dyneinopathies.
27The three nicotinamide mononucleotide adenylyltransferase (NMNAT) family members 28 synthesize the electron carrier nicotinamide adenine dinucleotide (NAD + ) and are essential for 29 cellular metabolism. In mammalian axons, NMNAT activity appears to be required for axon 30 survival and is predominantly provided by NMNAT2. NMNAT2 has recently been shown to also 31 function as a chaperone to aid in the refolding of misfolded proteins. Nmnat2 deficiency in 32 mice, or in its ortholog dNmnat in Drosophila, results in axon outgrowth and survival defects. 33Peripheral nerve axons in NMNAT2-deficient mice fail to extend and innervate targets, and 34 skeletal muscle is severely underdeveloped. In addition, removing NMNAT2 from established 35 axons initiates axon death by Wallerian degeneration. We report here on two stillborn siblings 36 with fetal akinesia deformation sequence (FADS), severely reduced skeletal muscle mass and 37 hydrops fetalis. Clinical exome sequencing identified compound heterozygous NMNAT2 38 variant alleles in both cases. Both protein variants are incapable of supporting axon survival in 39 mouse primary neuron cultures when overexpressed. In vitro assays demonstrate altered 40 protein stability and/or defects in NAD + synthesis and chaperone functions. Thus, both patient 41 NMNAT2 alleles are null or severely hypo-morphic. These data indicate a previously unknown 42 role for NMNAT2 in human neurological development and provide the first direct molecular 43 evidence to support the involvement of Wallerian degeneration in a human axonal disorder. 44 45 46 47 Fetal Akinesia Deformation Sequence (FADS) defines a broad range of disorders unified by 51 absent fetal movement resulting in secondary defects often leading to stillbirth or limited 52 postnatal survival 1; 2 . These secondary features include edema, hydrops fetalis, craniofacial 53 anomalies including micrognathia, lung hypoplasia, rocker bottom feet, intrauterine growth 54 restriction, and decreased muscle mass 3 . Through previous experimental models of fetal 55 paralysis, the secondary findings have been shown to be primarily caused by a lack of fetal 56 movement 1; 4 . FADS has both genetic and environmental causes that can affect any aspect of 57 the motor system including the central nervous system (CNS), peripheral nervous system 58 (PNS), neuromuscular junction (NMJ), and/or skeletal muscle. Although most cases of FADS 59 do not have a genetic diagnosis, multiple monogenic causes of FADS affecting PNS 60 innervation development have been identified to date including RAPSN, DOK7, MUSK 5-7 . 61 62Through whole exome sequencing and subsequent Sanger sequencing of a family with two 63 fetuses with FADS, we identified compound heterozygous mutations in a gene previously 64 unlinked to FADS, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2). NMNAT 65 family members were first shown to play a role in axon degeneration with the discovery of the 66 slow Wallerian Degeneration (Wld S ) mutant mouse that showed delayed axon degenera...
The Glycosylphosphatidylinositol (GPI) anchor is a post-translational modification added to approximately 150 different proteins to facilitate proper membrane anchoring and trafficking to lipid rafts.Biosynthesis and remodeling of the GPI anchor requires the activity of over twenty distinct genes. Defects in the biosynthesis of GPI anchors in humans leads to Inherited Glycosylphosphatidylinositol Deficiency (IGD). IGD patients display a wide range of phenotypes though the central nervous system (CNS) appears to be the most commonly affected tissue. A full understanding of the etiology of these phenotypes has been hampered by the lack of animal models due to embryonic lethality of GPI biosynthesis gene null mutants. Here we model IGD by genetically ablating GPI production in the CNS with a conditional mouse allele of phosphatidylinositol glycan anchor biosynthesis, class A (Piga) andNestin-Cre. We find that the mutants do not have structural brain defects but do not survive past weaning.The mutants show progressive decline with severe ataxia consistent with defects in cerebellar development. We show the mutants have reduced myelination and defective Purkinje cell development.Surprisingly we found Piga was expressed in a fairly restricted pattern in the early postnatal brain consistent with the defects we observed in our model. Thus, we have generated a novel mouse model of the neurological defects of IGD which demonstrates a critical role for GPI biosynthesis in cerebellar and white matter development.
The separate and concurrent effects of nutritional muscular dystrophy, induced by vitamin E-deficiency, and of denervation atrophy on wet weight, noncollagen protein nitrogen and deoxyribonucleic acid content, and on the activities of six acid hydrolases in the hamstring muscles of rabbits have been investigated. There are similarities in the patterns of increased acid hydrolase activity in both conditions, although in nutritional dystrophy the increments are much greater. Loss of muscle mass and noncollagen protein nitrogen content are much greater in denervation atrophy than in nutritional muscular dystrophy, whereas only in the latter condition is there a marked increase in deoxyribonucleic acid content. The effects of both conditions occurring simultaneously appear to be additive. The results also suggest that the increased activities of hydrolytic enzymes represent a common enzymatic mechanism that is involved in the loss of muscle mass and that most of the increments in hydrolase activity of dystrophic muscle originate from the pathological muscle cells themselves and may partially reflect attempts at regeneration.
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