PMC

Facial dysmorphology. Proband photographs of PI.p1, CF.p1, CF.p2, MA.p1, NN1.p1, and CC.p1. Clinical similarities include low hairline (especially European-descent patients), large ears with thick helices, thick eyebrows with synophrys deep-set eyes, strabismus, large nose with high nasal bridge (for subset), short and flat philtrum, large mouth with thinner upper lip and thicker everted lower lip, inferior/widely spaced teeth (PI.p1, CF.p1, CF.p2, NN1.p1), and tendency for a protruded tongue. Consent for the publication of photographs was obtained for these patients

TANC2 expression in human developing brain. a T-stochastic neighbor embedding of 4621 single-cell RNA sequencing (scRNA-seq) profiles from developing human brain samples identifies the major cell types in the developing brain. Cluster numbers are drawn directly from the source study, and biological interpretations can be found in Supplementary Table . b Violin plot showing TANC2 expression across the major cell types identified in the scRNA-seq (a). Samples are ordered according to the average expression level across single cells of each type, and p value represents Bonferroni-corrected p value quantified using Wilcoxon rank sum test. n.s.—p > 0.05. Underlying data are provided as a Source Data file

rols is enriched in glia and glial-specific knockdown of rols leads to increased courtship display frequency in adult flies. a Pan-neuronal staining (ELAV) of rols^{MI02479-TG4.1}>UAS-RedStinger, UAS-FLP, Ubi-p63E(FRT.STOP)Stinger third instar larva reveals that rols is not expressed in neurons. Scale bar = 100 μm. b Neuronal (ELAV) and glial (REPO) staining of adult fly brains using rols^{MI02479-TG4.1} > UAS-nls::GFP reveals that rols is expressed in a subset of glia (yellow arrows, and inset on right panel). Scale bar = 100 μm. c–f Glial-specific (Repo-Gal4) knockdown of rols (IR1, n = 16; IR2, n = 24) leads to increased courtship display duration when compared to controls (LacZ, n = 13; +, n = 15; IR1/+, n = 15; IR2/+, n = 14) (c, d) while copulation duration and success remained unchanged (e, f). Statistical results for d–f. ****p < 0.0001, ***p < 0.001, *p < 0.05; n.s. not significant. Error bars represent SEM. Underlying data are provided as a Source Data file

rols is an essential gene expressed in the developing and adult nervous system of flies. a Schematic of the rols locus where the rols^{MI02479-TG4.1} allele was generated by genetic conversion of the y¹ w^* Mi{MIC}rols^MI02479 allele via RMCE using ΦC31 expression to swap the original MiMIC insertion cassette for an SA-T2A-GAL4-polyA. The SA-T2A-GAL4 in the first intron of rols acts as an artificial exon resulting in early truncation of the rols transcript due to the polyA sequence. During translation, the T2A promotes ribosomal skipping and subsequent translation of GAL4 under the endogenous regulatory elements of rols. b Homozygous rols^{MI02479-TG4.1} mutant flies are embryonic lethal. Embryonic lethality is also observed in rols^{MI02479-TG4.1}/Df(3L)ED4475, which can by rescued by introduction of a 80 kb genomic rescue construct inserted on chromosome 2 (VK37). c Larval and adult brain staining (GFP) of rols^{MI02479-TG4.1}>UAS-mCD8::GFP flies reveals membranous staining throughout the brain at both stages. Scale bar = 50 μm

rols is historically expressed in developing muscle cells and muscle-specific knockdown of rols increases satellite bouton number and decreases GluRIIA levels at the third instar larval NMJ. a G-TRACE analysis by crossing rols^{MI02479-TG4.1} to UAS-RedStinger, UAS-FLP, Ubi-p63E(FRT.STOP)Stinger reveals that Rols is currently expressed in wrapping glia at the NMJ (yellow arrow) and was historically expressed in muscle cells (white arrows). Scale bar = 50 μm. b, c Muscle-specific (C57-Gal4) knockdown of rols showed an increased number of satellite boutons (b) (C57/+, n = 19; C57>RNAi1, n = 26; C57>RNAi2, n = 23; C57-RES1, n = 16; C57-RES2 n = 21) and decreased normalized fluorescent intensity of GluRIIA (c) (C57/+, n = 25; C57>RNAi1, n = 25; C57>RNAi2, n = 28; C57-RES1, n = 14; C57-RES2 n = 13) compared to wild-type larvae. Scale bar = 10 μm. Underlying data are provided as a Source Data file. Statistical results for b and c. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05; n.s. not significant. Error bars represent SEM

Prioritizing ASD candidate genes based on gene intolerance metrics and enrichment of FMRP/RBFOX targets. a Burden of de novo LGD mutations in probands of SSC simplex quad families for three categories: (i) all de novo LGD events; (ii) de novo LGD events excluding genes where significant burden has been reached; and (iii) de novo LGD mutations in intolerant genes without significance. The error bars represents a 95% confidence interval for the mean rates. Underlying data are provided as a Source Data file. b Enrichment of genes with de novo LGD mutations in FMRP and RBFOX targets. Enrichment is performed after excluding genes that reached significance (top panel) and the same but requiring that the genes are intolerant to mutation (bottom panel). c Selection of genes for targeted sequencing. One hundred and twenty-eight intolerant genes were prioritized by pLI score (<0.84) and RVIS percentile (>32) from the SSC and ASC cohorts, of which 58 genes were FMRP/RBFOX targets (Table ). Fourteen genes were selected from the 58 genes for targeted sequencing in 2514 ASD probands from the ACGC cohort

Location distribution and transmission pattern of TANC2 mutations. a A protein domain graph (DOG) plot shows the positions of the 16 LGD (above) and 5 missense (bottom) mutations in TANC2. The annotated and predicted domains in TANC2 are presented. A potential missense cluster is identified at the carboxy terminus of the ATPase domain. b Microdeletions identified in four patients (PI.p1, DU.p1, CF.p1, CF.p2) from this study and one patient (322959) in the DECIPHER database. CF.p1 and CF.p2 are affected siblings as noted in c. c Pedigree plot of the five families with transmitted disruptive variants. The carrier father in family SS2 has been diagnosed with behavioral and neuropsychiatric disorders, including bipolar disorder, ADHD, PTSD, and social issues reflected by the adult SRS score. The carrier mother in family GU has ID and experienced seizures, motor delay, and learning difficulties in school especially during her teenage years. The carrier father in family NN2 is suspected to have ID and has a psychiatric disorder history. The carrier father from the family CF experienced delayed motor development, learning difficulties in school, and also suspected to have ID. No clinical assessment of detailed developmental or neuropsychiatric history was possible for carrier father in family TI