The actin cytoskeleton is regulated by many proteins including capping proteins that stabilize actin filaments (F-actin) by inhibiting actin polymerization and depolymerization. Here, we report two pediatric probands who carry damaging heterozygous de novo mutations in CAPZA2 (HGNC: 1490) and exhibit neurological symptoms with shared phenotypes including global motor development delay, speech delay, intellectual disability, hypotonia and a history of seizures. CAPZA2 encodes a subunit of an F-actin-capping protein complex (CapZ). CapZ is an obligate heterodimer consisting of α and β heterodimer conserved from yeast to human. Vertebrate genomes contain three α subunits encoded by three different genes and CAPZA2 encodes the α2 subunit. The single orthologue of CAPZA genes in Drosophila is cpa. Loss of cpa leads to lethality in early development and expression of the human reference; CAPZA2 rescues this lethality. However, the two CAPZA2 variants identified in the probands rescue this lethality at lower efficiency than the reference. Moreover, expression of the CAPZA2 variants affects bristle morphogenesis, a process that requires extensive actin polymerization and bundling during development. Taken together, our findings suggest that variants in CAPZA2 lead to a non-syndromic neurodevelopmental disorder in children.

The Roundabout (Robo) receptors, located on growth cones of neurons, induce axon repulsion in response to the extracellular ligand Slit. The Robo family of proteins controls midline crossing of commissural neurons during development in flies. Mono- and bi-allelic variants in human ROBO1 (HGNC: 10249) have been associated with incomplete penetrance and variable expressivity for a breath of phenotypes, including neurodevelopmental defects such as strabismus, pituitary defects, intellectual impairment, as well as defects in heart and kidney. Here, we report two novel ROBO1 variants associated with very distinct phenotypes. A homozygous missense p.S1522L variant in three affected siblings with nystagmus; and a monoallelic de novo p.D422G variant in a proband who presented with early-onset epileptic encephalopathy. We modeled these variants in Drosophila and first generated a null allele by inserting a CRIMIC T2A-GAL4 in an intron. Flies that lack robo1 exhibit reduced viability but have very severe midline crossing defects in the central nervous system. The fly wild-type cDNA driven by T2A-Gal4 partially rescues both defects. Overexpression of the human reference ROBO1 with T2A-GAL4 is toxic and reduces viability, whereas the recessive p.S1522L variant is less toxic, suggesting that it is a partial loss-of-function allele. In contrast, the dominant variant in fly robo1 (p.D413G) affects protein localization, impairs axonal guidance activity and induces mild phototransduction defects, suggesting that it is a neomorphic allele. In summary, our studies expand the phenotypic spectrum associated with ROBO1 variant alleles.

SUPT16H encodes the large subunit of the FAcilitate Chromatin Transcription (FACT) complex, which functions as a nucleosome organizer during transcription. We identified two individuals from unrelated families carrying de novo missense variants in SUPT16H. The probands exhibit global developmental delay, intellectual disability, epilepsy, facial dysmorphism and brain structural abnormalities. We used Drosophila to characterize two variants: p.T171I and p.G808R. Loss of the fly ortholog, dre4, causes lethality at an early developmental stage. RNAi-mediated knockdown of dre4 in either glia or neurons causes severely reduced eclosion and longevity. Tissue-specific knockdown of dre4 in the eye or wing leads to the loss of these tissues, whereas overexpression of SUPT16H has no dominant effect. Moreover, expression of the reference SUPT16H significantly rescues the loss-of-function phenotypes in the nervous system as well as wing and eye. In contrast, expression of SUPT16H p.T171I or p.G808R rescues the phenotypes poorly, indicating that the variants are partial loss-of-function alleles. While previous studies argued that the developmental arrest caused by loss of dre4 is due to impaired ecdysone production in the prothoracic gland, our data show that dre4 is required for proper cell growth and survival in multiple tissues in a cell-autonomous manner. Altogether, our data indicate that the de novo loss-of-function variants in SUPT16H are indeed associated with developmental and neurological defects observed in the probands.