Biallelic hypomorphic mutations in HEATR5B, encoding HEAT repeat-containing protein 5B, in a neurological syndrome with pontocerebellar hypoplasia.

HEAT repeats are 37-47 amino acid flexible tandem repeat structural motifs occurring in a wide variety of eukaryotic proteins with diverse functions. Due to their ability to undergo elastic conformational changes, they often serve as scaffolds at sites of protein interactions. Here, we describe four affected children from two families presenting with pontocerebellar hypoplasia manifest clinically with neonatal seizures, severe intellectual disability, and motor delay. Whole exome sequencing identified biallelic variants at predicted splice sites in intron 31 of HEATR5B, encoding the HEAT repeat-containing protein 5B segregating in a recessive fashion. Aberrant splicing was found in patient fibroblasts, which correlated with reduced levels of HEATR5B protein. HEATR5B is expressed during brain development in human, and we failed to recover live-born homozygous Heatr5b knockout mice. Taken together, our results implicate loss of HEATR5B in pontocerebellar hypoplasia.

A relatively common homozygous TRAPPC4 splicing variant is associated with an early-infantile neurodegenerative syndrome.

Trafficking protein particle (TRAPP) complexes, which include the TRAPPC4 protein, regulate membrane trafficking between lipid organelles in a process termed vesicular tethering. TRAPPC4 was recently implicated in a recessive neurodevelopmental condition in four unrelated families due to a shared c.454+3A>G splice variant. Here, we report 23 patients from 17 independent families with an early-infantile-onset neurodegenerative presentation, where we also identified the homozygous variant hg38:11:119020256 A>G (NM_016146.5:c.454+3A>G) in TRAPPC4 through exome or genome sequencing. No other clinically relevant TRAPPC4 variants were identified among any of over 10,000 patients with neurodevelopmental conditions. We found the carrier frequency of TRAPPC4 c.454+3A>G was 2.4-5.4 per 10,000 healthy individuals. Affected individuals with the homozygous TRAPPC4 c.454+3A>G variant showed profound psychomotor delay, developmental regression, early-onset epilepsy, microcephaly and progressive spastic tetraplegia. Based upon RNA sequencing, the variant resulted in partial exon 3 skipping and generation of an aberrant transcript owing to use of a downstream cryptic splice donor site, predicting a premature stop codon and nonsense mediated decay. These data confirm the pathogenicity of the TRAPPC4 c.454+3A>G variant, and refine the clinical presentation of TRAPPC4-related encephalopathy.

Biallelic variants in HPDL, encoding 4-hydroxyphenylpyruvate dioxygenase-like protein, lead to an infantile neurodegenerative condition.

PURPOSE: Dioxygenases are oxidoreductase enzymes with roles in metabolic pathways necessary for aerobic life. 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL), encoded by HPDL, is an orphan paralogue of 4-hydroxyphenylpyruvate dioxygenase (HPD), an iron-dependent dioxygenase involved in tyrosine catabolism. The function and association of HPDL with human diseases remain unknown. METHODS: We applied exome sequencing in a cohort of over 10,000 individuals with neurodevelopmental diseases. Effects of HPDL loss were investigated in vitro and in vivo, and through mass spectrometry analysis. Evolutionary analysis was performed to investigate the potential functional separation of HPDL from HPD. RESULTS: We identified biallelic variants in HPDL in eight families displaying recessive inheritance. Knockout mice closely phenocopied humans and showed evidence of apoptosis in multiple cellular lineages within the cerebral cortex. HPDL is a single-exonic gene that likely arose from a retrotransposition event at the base of the tetrapod lineage, and unlike HPD, HPDL is mitochondria-localized. Metabolic profiling of HPDL mutant cells and mice showed no evidence of altered tyrosine metabolites, but rather notable accumulations in other metabolic pathways. CONCLUSION: The mitochondrial localization, along with its disrupted metabolic profile, suggests HPDL loss in humans links to a unique neurometabolic mitochondrial infantile neurodegenerative condition.

Loss of NARS1 impairs progenitor proliferation in cortical brain organoids and leads to microcephaly.

Asparaginyl-tRNA synthetase1 (NARS1) is a member of the ubiquitously expressed cytoplasmic Class IIa family of tRNA synthetases required for protein translation. Here, we identify biallelic missense and frameshift mutations in NARS1 in seven patients from three unrelated families with microcephaly and neurodevelopmental delay. Patient cells show reduced NARS1 protein, impaired NARS1 activity and impaired global protein synthesis. Cortical brain organoid modeling shows reduced proliferation of radial glial cells (RGCs), leading to smaller organoids characteristic of microcephaly. Single-cell analysis reveals altered constituents of both astrocytic and RGC lineages, suggesting a requirement for NARS1 in RGC proliferation. Our findings demonstrate that NARS1 is required to meet protein synthetic needs and to support RGC proliferation in human brain development.

Motoneuron expression profiling identifies an association between an axonal splice variant of HDGF-related protein 3 and peripheral myelination.

Disorders that disrupt myelin formation during development or in adulthood, such as multiple sclerosis and peripheral neuropathies, lead to severe pathologies, illustrating myelin's crucial role in normal neural functioning. However, although our understanding of glial biology is increasing, the signals that emanate from axons and regulate myelination remain largely unknown. To identify the core components of the myelination process, here we adopted a microarray analysis approach combined with laser-capture microdissection of spinal motoneurons during the myelinogenic phase of development. We identified neuronal genes whose expression was enriched during myelination and further investigated hepatoma-derived growth factor-related protein 3 (HRP3 or HDGFRP3). HRP3 was strongly expressed in the white matter fiber tracts of the peripheral (PNS) and central (CNS) nervous systems during myelination and remyelination in a cuprizone-induced demyelination model. The dynamic localization of HPR3 between axons and nuclei during myelination was consistent with its axonal localization during neuritogenesis. To study this phenomenon, we identified two splice variants encoded by the HRP3 gene: the canonical isoform HRP3-I and a newly recognized isoform, HRP3-II. HRP3-I remained solely in the nucleus, whereas HRP3-II displayed distinct axonal localization both before and during myelination. Interestingly, HRP3-II remained in the nuclei of unmyelinated neurons and glial cells, suggesting the existence of a molecular machinery that transfers it to and retains it in the axons of neurons fated for myelination. Overexpression of HRP3-II, but not of HRP3-I, increased Schwann cell numbers and myelination in PNS neuron-glia co-cultures. However, HRP3-II overexpression in CNS co-cultures did not alter myelination.

Pathogenic ARH3 mutations result in ADP-ribose chromatin scars during DNA strand break repair.

Neurodegeneration is a common hallmark of individuals with hereditary defects in DNA single-strand break repair; a process regulated by poly(ADP-ribose) metabolism. Recently, mutations in the ARH3 (ADPRHL2) hydrolase that removes ADP-ribose from proteins have been associated with neurodegenerative disease. Here, we show that ARH3-mutated patient cells accumulate mono(ADP-ribose) scars on core histones that are a molecular memory of recently repaired DNA single-strand breaks. We demonstrate that the ADP-ribose chromatin scars result in reduced endogenous levels of important chromatin modifications such as H3K9 acetylation, and that ARH3 patient cells exhibit measurable levels of deregulated transcription. Moreover, we show that the mono(ADP-ribose) scars are lost from the chromatin of ARH3-defective cells in the prolonged presence of PARP inhibition, and concomitantly that chromatin acetylation is restored to normal. Collectively, these data indicate that ARH3 can act as an eraser of ADP-ribose chromatin scars at sites of PARP activity during DNA single-strand break repair.

Loss of the neural-specific BAF subunit ACTL6B relieves repression of early response genes and causes recessive autism.

Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such "early activation" genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.

Recurrent homozygous damaging mutation in TMX2, encoding a protein disulfide isomerase, in four families with microlissencephaly.

BACKGROUND: Protein disulfide isomerase (PDI) proteins are part of the thioredoxin protein superfamily. PDIs are involved in the formation and rearrangement of disulfide bonds between cysteine residues during protein folding in the endoplasmic reticulum and are implicated in stress response pathways. METHODS: Eight children from four consanguineous families residing in distinct geographies within the Middle East and Central Asia were recruited for study. All probands showed structurally similar microcephaly with lissencephaly (microlissencephaly) brain malformations. DNA samples from each family underwent whole exome sequencing, assessment for repeat expansions and confirmatory segregation analysis. RESULTS: An identical homozygous variant in TMX2 (c.500G>A), encoding thioredoxin-related transmembrane protein 2, segregated with disease in all four families. This variant changed the last coding base of exon 6, and impacted mRNA stability. All patients presented with microlissencephaly, global developmental delay, intellectual disability and epilepsy. While TMX2 is an activator of cellular C9ORF72 repeat expansion toxicity, patients showed no evidence of C9ORF72 repeat expansions. CONCLUSION: The TMX2 c.500G>A allele associates with recessive microlissencephaly, and patients show no evidence of C9ORF72 expansions. TMX2 is the first PDI implicated in a recessive disease, suggesting a protein isomerisation defect in microlissencephaly.

Biallelic Mutations in ADPRHL2, Encoding ADP-Ribosylhydrolase 3, Lead to a Degenerative Pediatric Stress-Induced Epileptic Ataxia Syndrome.

ADP-ribosylation, the addition of poly-ADP ribose (PAR) onto proteins, is a response signal to cellular challenges, such as excitotoxicity or oxidative stress. This process is catalyzed by a group of enzymes referred to as poly(ADP-ribose) polymerases (PARPs). Because the accumulation of proteins with this modification results in cell death, its negative regulation restores cellular homeostasis: a process mediated by poly-ADP ribose glycohydrolases (PARGs) and ADP-ribosylhydrolase proteins (ARHs). Using linkage analysis and exome or genome sequencing, we identified recessive inactivating mutations in ADPRHL2 in six families. Affected individuals exhibited a pediatric-onset neurodegenerative disorder with progressive brain atrophy, developmental regression, and seizures in association with periods of stress, such as infections. Loss of the Drosophila paralog Parg showed lethality in response to oxidative challenge that was rescued by human ADPRHL2, suggesting functional conservation. Pharmacological inhibition of PARP also rescued the phenotype, suggesting the possibility of postnatal treatment for this genetic condition.