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.

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.

The three nicotinamide mononucleotide adenylyltransferase (NMNAT) family members synthesize the electron carrier nicotinamide adenine dinucleotide (NAD ⁺ ) and are essential for cellular metabolism. In mammalian axons, NMNAT activity appears to be required for axon survival and is predominantly provided by NMNAT2. NMNAT2 has recently been shown to also function as a chaperone to aid in the refolding of misfolded proteins. Nmnat2 deficiency in mice, or in its ortholog dNmnat in Drosophila , results in axon outgrowth and survival defects. Peripheral nerve axons in NMNAT2-deficient mice fail to extend and innervate targets, and skeletal muscle is severely underdeveloped. In addition, removing NMNAT2 from established axons initiates axon death by Wallerian degeneration. We report here on two stillborn siblings with fetal akinesia deformation sequence (FADS), severely reduced skeletal muscle mass and hydrops fetalis. Clinical exome sequencing identified compound heterozygous NMNAT2 variant alleles in both cases. Both protein variants are incapable of supporting axon survival in mouse primary neuron cultures when overexpressed. In vitro assays demonstrate altered protein stability and/or defects in NAD ⁺ synthesis and chaperone functions. Thus, both patient NMNAT2 alleles are null or severely hypo-morphic. These data indicate a previously unknown role for NMNAT2 in human neurological development and provide the first direct molecular evidence to support the involvement of Wallerian degeneration in a human axonal disorder.

Primary ciliary dyskinesia (PCD) is a human condition of dysfunctional motile cilia characterized by recurrent lung infection, infertility, organ laterality defects, and partially penetrant hydrocephalus. We recovered a mouse mutant from a forward genetic screen that developed all the phenotypes of PCD. Whole exome sequencing identified this primary ciliary dyskinesia only (Pcdo) allele to be a nonsense mutation (c.5236A>T) in the Spag17 coding sequence creating a premature stop codon at position 1746 (K1746*). The Pcdo variant abolished different isoforms of SPAG17 in the Pcdo mutant testis but not in the brain. Our data indicate differential requirements for SPAG17 in different motile cilia cell types. SPAG17 is required for proper development of the sperm flagellum, and is essential for either development or stability of the C1 microtubule structure within cilia, but not the brain ependymal cilia. We identified changes in ependymal cilia beating frequency but these did not apparently alter lateral ventricle cerebrospinal fluid (CSF) flow. Aqueductal (Aq) stenosis resulted in significantly slower and abnormally directed CSF flow and we suggest this is the root cause of the hydrocephalus. The Spag17 ^Pcdo homozygous mutant mice are generally viable to adulthood, but have a significantly shortened life span with chronic morbidity. Our data indicate that the c.5236A>T Pcdo variant is a hypomorphic allele of Spag17 gene that causes phenotypes related to motile, but not primary, cilia. Spag17 ^Pcdo is a novel and useful model for elucidating the molecular mechanisms underlying development of PCD in the mouse.

The glycosylphosphatidylinositol (GPI) anchor attaches nearly 150 proteins to the cell surface. Patients with pathogenic variants in GPI biosynthetic pathway genes display an array of phenotypes including seizures, developmental delay, dysmorphic facial features and cleft palate. There is virtually no mechanism to explain these phenotypes. we identified a novel mouse mutant ( cleft lip/palate, edema and exencephaly; Clpex ) with a hypomorphic mutation in Post-Glycophosphatidylinositol Attachment to Proteins-2 (Pgap2). Pgap2 is one of the final proteins in the GPI biosynthesis pathway and is required for anchor maturation. We found the Clpex mutation results in a global decrease in surface GPI expression. Surprisingly, Pgap2 showed tissue specific expression with enrichment in the affected tissues of the Clpex mutant. We found the phenotype in Clpex mutants is due to apoptosis of neural crest cells (NCCs) and the cranial neuroepithelium, as is observed in the GPI anchored Folate Receptor 1 ^-/- mouse. We showed folinic acid supplementation in utero can rescue the cleft lip phenotype in Clpex . Finally, we generated a novel mouse model of NCC-specific total GPI deficiency in the Wnt1-Cre lineage. These mutants developed median cleft lip and palate demonstrating a cell autonomous role for GPI biosynthesis in NCC development.