三谷 忠宏

The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential subunits for guanine nucleotide exchange factor activity for Rab1 GTPase. Pathogenic variants in specific TRAPP subunits are associated with neurological disorders. We undertook exome sequencing in three unrelated families of Caucasian, Turkish and French-Canadian ethnicities with seven affected children that showed features of early-onset seizures, developmental delay, microcephaly, sensorineural deafness, spastic quadriparesis and progressive cortical and cerebellar atrophy in an effort to determine the genetic aetiology underlying neurodevelopmental disorders. All seven affected subjects shared the same identical rare, homozygous, potentially pathogenic variant in a non-canonical, well-conserved splice site within TRAPPC4 (hg19:chr11:g.118890966A>G; TRAPPC4: NM_016146.5; c.454+3A>G). Single nucleotide polymorphism array analysis revealed there was no haplotype shared between the tested Turkish and Caucasian families suggestive of a variant hotspot region rather than a founder effect. In silico analysis predicted the variant to cause aberrant splicing. Consistent with this, experimental evidence showed both a reduction in full-length transcript levels and an increase in levels of a shorter transcript missing exon 3, suggestive of an incompletely penetrant splice defect. TRAPPC4 protein levels were significantly reduced whilst levels of other TRAPP complex subunits remained unaffected. Native polyacrylamide gel electrophoresis and size exclusion chromatography demonstrated a defect in TRAPP complex assembly and/or stability. Intracellular trafficking through the Golgi using the marker protein VSVG-GFP-ts045 demonstrated significantly delayed entry into and exit from the Golgi in fibroblasts derived from one of the affected subjects. Lentiviral expression of wild-type TRAPPC4 in these fibroblasts restored trafficking, suggesting that the trafficking defect was due to reduced TRAPPC4 levels. Consistent with the recent association of the TRAPP complex with autophagy, we found that the fibroblasts had a basal autophagy defect and a delay in autophagic flux, possibly due to unsealed autophagosomes. These results were validated using a yeast trs23 temperature sensitive variant that exhibits constitutive and stress-induced autophagic defects at permissive temperature and a secretory defect at restrictive temperature. In summary we provide strong evidence for pathogenicity of this variant in a member of the core TRAPP subunit, TRAPPC4 that associates with vesicular trafficking and autophagy defects. This is the first report of a TRAPPC4 variant, and our findings add to the growing number of TRAPP-associated neurological disorders.

Lissencephaly comprises a spectrum of malformations of cortical development. This spectrum includes agyria, pachygyria, and subcortical band heterotopia; each represents anatomical malformations of brain cortical development caused by neuronal migration defects. The molecular etiologies of neuronal migration anomalies are highly enriched for genes encoding microtubules and microtubule-associated proteins, and this enrichment highlights the critical role for these genes in cortical growth and gyrification. Using exome sequencing and family based rare variant analyses, we identified a homozygous variant (c.997C>T [p.Arg333Cys]) in TUBGCP2, encoding gamma-tubulin complex protein 2 (GCP2), in two individuals from a consanguineous family; both individuals presented with microcephaly and developmental delay. GCP2 forms the multiprotein γ-tubulin ring complex (γ-TuRC) together with γ-tubulin and other GCPs to regulate the assembly of microtubules. By querying clinical exome sequencing cases and through GeneMatcher-facilitated collaborations, we found three additional families with bi-allelic variation and similarly affected phenotypes including a homozygous variant (c.1843G>C [p.Ala615Pro]) in two families and compound heterozygous variants consisting of one missense variant (c.889C>T [p.Arg297Cys]) and one splice variant (c.2025-2A>G) in another family. Brain imaging from all five affected individuals revealed varying degrees of cortical malformations including pachygyria and subcortical band heterotopia, presumably caused by disruption of neuronal migration. Our data demonstrate that pathogenic variants in TUBGCP2 cause an autosomal recessive neurodevelopmental trait consisting of a neuronal migration disorder, and our data implicate GCP2 as a core component of γ-TuRC in neuronal migrating cells.

NTNG2 encodes netrin-G2, a membrane-anchored protein implicated in the molecular organization of neuronal circuitry and synaptic organization and diversification in vertebrates. In this study, through a combination of exome sequencing and autozygosity mapping, we have identified 16 individuals (from seven unrelated families) with ultra-rare homozygous missense variants in NTNG2; these individuals present with shared features of a neurodevelopmental disorder consisting of global developmental delay, severe to profound intellectual disability, muscle weakness and abnormal tone, autistic features, behavioral abnormalities, and variable dysmorphisms. The variants disrupt highly conserved residues across the protein. Functional experiments, including in silico analysis of the protein structure, in vitro assessment of cell surface expression, and in vitro knockdown, revealed potential mechanisms of pathogenicity of the variants, including loss of protein function and decreased neurite outgrowth. Our data indicate that appropriate expression of NTNG2 plays an important role in neurotypical development.

Single transcription factors (TFs) regulate multiple developmental pathways, but the underlying mechanisms remain unclear. Here, we quantitatively characterized the genome-wide occupancy profiles of BLIMP1, a key transcriptional regulator for diverse developmental processes, during the development of three germ-layer derivatives (photoreceptor precursors, embryonic intestinal epithelium and plasmablasts) and the germ cell lineage (primordial germ cells). We identified BLIMP1-binding sites shared among multiple developmental processes, and such sites were highly occupied by BLIMP1 with a stringent recognition motif and were located predominantly in promoter proximities. A subset of bindings common to all the lineages exhibited a new, strong recognition sequence, a GGGAAA repeat. Paradoxically, however, the shared/common bindings had only a slight impact on the associated gene expression. In contrast, BLIMP1 occupied more distal sites in a cell type-specific manner; despite lower occupancy and flexible sequence recognitions, such bindings contributed effectively to the repression of the associated genes. Recognition motifs of other key TFs in BLIMP1-binding sites had little impact on the expression-level changes. These findings suggest that the shared/common sites might serve as potential reservoirs of BLIMP1 that functions at the specific sites, providing the foundation for a unified understanding of the genome regulation by BLIMP1, and, possibly, TFs in general.

Germ cell specification is accompanied by epigenetic remodeling, the scale and specificity of which are unclear. Here, we quantitatively delineate chromatin dynamics during induction of mouse embryonic stem cells (ESCs) to epiblast-like cells (EpiLCs) and from there into primordial germ cell-like cells (PGCLCs), revealing large-scale reorganization of chromatin signatures including H3K27me3 and H3K9me2 patterns. EpiLCs contain abundant bivalent gene promoters characterized by low H3K27me3, indicating a state primed for differentiation. PGCLCs initially lose H3K4me3 from many bivalent genes but subsequently regain this mark with concomitant upregulation of H3K27me3, particularly at developmental regulatory genes. PGCLCs progressively lose H3K9me2, including at lamina-associated perinuclear heterochromatin, resulting in changes in nuclear architecture. T recruits H3K27ac to activate BLIMP1 and early mesodermal programs during PGCLC specification, which is followed by BLIMP1-mediated repression of a broad range of targets, possibly through recruitment and spreading of H3K27me3. These findings provide a foundation for reconstructing regulatory networks of the germline epigenome.

A stroke-like episode is a core symptom in mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Proton magnetic resonance spectroscopy (1H-MRS) is useful in the diagnosis of mitochondrial diseases. We report an 8-year-old girl with MELAS, presenting with a seizure and blindness. 1H-MRS showed a strikingly elevated lactate peak in the right occipital region, where no abnormal signals appeared on either T2-W or diffusion-weighted MRI. She recovered completely within a day. We describe this mild clinical condition with abnormal lactate peak in normal-appearing gray matter as a transient ischemic attack-like episode in MELAS.

We report on an 8-year-old boy with non-paraneoplastic anti-NMDA receptor (NMDAR) encephalitis, who presented with psychotic symptoms and involuntary movement following an intractable seizure. His serum and CSF tested positive for anti-NMDAR antibodies. He received an initial immunotherapy consisting of methylprednisolone pulse therapy (mPSL) and intravenous immunoglobulin therapy (IVIg), without any clinical improvement. He had three cycles of monthly cyclophosphamide pulse therapy (500 mg/m2), and his clinical condition started to improve gradually two weeks after the first cycle, without any side effects. Six months after onset, he tested normal upon standard neurological examination. Cyclophosphamide therapy should be considered for children with anti-NMDAR encephalitis, as well as mPSL and IVIg.

The changes in the signals for brain metabolites in the left cerebellum of a 14-year-old boy with acute hemicerebellitis were monitored using proton magnetic resonance spectroscopy (MRS). From the onset of disease treatment to long-term follow-up, MRS data were serially acquired from the left and right cerebella, basal ganglia (BG), and centrum semiovale (CS). Large fluctuations in his MRS signals were observed in the left cerebellum. At onset (first day), his glutamate/glutamine complex signals were increased (>mean ± 2 standard deviations [SD] of the control), and those for N-acetylaspartate/N-acetylaspartylglutamate and myo-inositol were decreased (<2SD). By the 25th day, these signals had recovered to normal levels, while those for choline (Cho) were increased. In other locations, the signals for mIns in the BG and Cho in the CS were decreased on the seventh day. By the 201st day, the levels of all metabolites in all locations had recovered to within ± 2SD of the control levels. In vivo proton MRS monitoring demonstrated reversible metabolite changes associated with acute hemicerebellitis, which should contribute to its differential diagnosis from brain tumors.