Mutations in NCAPG2 Cause a Severe Neurodevelopmental Syndrome that Expands the Phenotypic Spectrum of Condensinopathies

Abstract

The use of whole-exome and whole-genome sequencing has been a catalyst for a genotype-first approach to diagnostics. Under this paradigm, we have implemented systematic sequencing of neonates and young children with a suspected genetic disorder. Here, we report on two families with recessive mutations in NCAPG2 and overlapping clinical phenotypes that include severe neurodevelopmental defects, failure to thrive, ocular abnormalities, and defects in urogenital and limb morphogenesis. NCAPG2 encodes a member of the condensin II complex, necessary for the condensation of chromosomes prior to cell division. Consistent with a causal role for NCAPG2, we found abnormal chromosome condensation, augmented anaphase chromatin-bridge formation, and micronuclei in daughter cells of proband skin fibroblasts. To test the functional relevance of the discovered variants, we generated an ncapg2 zebrafish model. Morphants displayed clinically relevant phenotypes, such as renal anomalies, microcephaly, and concomitant increases in apoptosis and altered mitotic progression. These could be rescued by wild-type but not mutant human NCAPG2 mRNA and were recapitulated in CRISPR-Cas9 F0 mutants. Finally, we noted that the individual with a complex urogenital defect also harbored a heterozygous NPHP1 deletion, a common contributor to nephronophthisis. To test whether sensitization at the NPHP1 locus might contribute to a more severe renal phenotype, we co-suppressed nphp1 and ncapg2, which resulted in significantly more dysplastic renal tubules in zebrafish larvae. Together, our data suggest that impaired function of NCAPG2 results in a severe condensinopathy, and they highlight the potential utility of examining candidate pathogenic lesions beyond the primary disease locus.We cultured primary dermal fibroblasts in DMEM (Sigma Aldrich), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin. We synchronized cells by reducing serum content to 0.1% for 24 hr, then by stimulating the cells for 24 hr with 10% FBS. 19

Bi-allelic Variants in DYNC1I2 Cause Syndromic Microcephaly with Intellectual Disability, Cerebral Malformations, and Dysmorphic Facial Features

“…Additionally, we measured the size of the optic tecta, neuron-rich sensory processing centers in the zebrafish brain whose size correlates with overall head size. 39,44,49 Consistent with our bright-field dorsal head-size measurements, we observed a significant reduction in the area of the optic tecta in dync1i2a morphants (p < 0.01 versus controls), but the size of these structures was restored significantly when MO was coinjected with WT DYNC1I2 mRNA (p < 0.01 versus MO alone; Figures S7A and S7C). Overall, we observed marked disorganization of axonal patterning in the zebrafish head.…”

“…Heteroduplex analysis, cloning, and direct sequencing of DNA derived from single embryos (one control, five sgRNA þ Cas9-injected embryos, with 16 clones per embryo at 1 dpf) resulted in 70%, 84%, and 50% estimated mosaicism, respectively ( Figures S3A, S3B, S3C, S4A, S4B, and S4C). We have shown previously that zebrafish have robust anatomical surrogates for head size and craniofacial patterning, 33,[35][36][37][38][39][40]46 phenotypes that we can capture simultaneously in a semi-automated fashion by using a capillary-based zebrafish imaging platform. 47 We injected embryo batches with the dync1i2a or dync1i2b sgRNAs with or without Cas9, reared animals to 3 dpf, and acquired dorsal bright-field images to measure head size and ventral fluorescent images of GFP-positive cells painting the pharyngeal skeleton.…”

“…[33][34][35][36][37] TUNEL Assay and Whole-Mount Immunostaining TUNEL was performed essentially as described. 33,[37][38][39] In brief, we dechorionated embryos at 2 dpf and fixed them in 4% paraformaldehyde (PFA) overnight at 4 C. They were dehydrated in 100% methanol for 2 h at À20 C prior to rehydration and permeabilization with proteinase K for 10 min and post fixation in 4% PFA for 20 min. Embryos were treated with equilibration buffer for 1 h and incubated overnight in TdT enzyme at 4 C. Embryos were then treated with the digoxigenin provided in the ApopTag red in situ apoptosis detection kit (Millipore) for 2 h and washed three times for 10 min each with PBST.…”

“…Immunostaining was performed as described. 33,[37][38][39][40] Dechorionated embryos at 2 dpf (pH3); and embryos at 1 dpf or larvae at 3 dpf (acetylated a-tubulin) were fixed overnight in 4% PFA (pH3) or Dent's solution (80% methanol, 40% DMSO; acetylated tubulin), respectively, dehydrated in methanol, and rehydrated gradually with PBST in methanol. We bleached for 10 min in 9mL PBST þ 1mL H 2 O 2 þ 0.05 g KOH and washed three times for 10 min each in PBST.…”

“…We wondered whether apoptosis could account for the reduction in head size of dync1i2a zebrafish models, as had been shown previously for other models of microcephaly. 33,[37][38][39]50,51 To test this, we performed TUNEL staining in embryos at 2 dpf and quantified TUNEL-positive cells within a normalized area of the dorsal forebrain between the eyes. In dync1i2a morphants, we observed a significant increase in cell death that could be rescued with co-injection of WT human DYNC1I2 mRNA (p < 0.0001, MO versus control; p < 0.01, MO versus MOþWT mRNA; Figures 4A and 4B).…”