Dual Molecular Effects of Dominant RORA Mutations Cause Two Variants of Syndromic Intellectual Disability with Either Autism or Cerebellar Ataxia.

RORα, the RAR-related orphan nuclear receptor alpha, is essential for cerebellar development. The spontaneous mutant mouse staggerer, with an ataxic gait caused by neurodegeneration of cerebellar Purkinje cells, was discovered two decades ago to result from homozygous intragenic Rora deletions. However, RORA mutations were hitherto undocumented in humans. Through a multi-centric collaboration, we identified three copy-number variant deletions (two de novo and one dominantly inherited in three generations), one de novo disrupting duplication, and nine de novo point mutations (three truncating, one canonical splice site, and five missense mutations) involving RORA in 16 individuals from 13 families with variable neurodevelopmental delay and intellectual disability (ID)-associated autistic features, cerebellar ataxia, and epilepsy. Consistent with the human and mouse data, disruption of the D. rerio ortholog, roraa, causes significant reduction in the size of the developing cerebellum. Systematic in vivo complementation studies showed that, whereas wild-type human RORA mRNA could complement the cerebellar pathology, missense variants had two distinct pathogenic mechanisms of either haploinsufficiency or a dominant toxic effect according to their localization in the ligand-binding or DNA-binding domains, respectively. This dichotomous direction of effect is likely relevant to the phenotype in humans: individuals with loss-of-function variants leading to haploinsufficiency show ID with autistic features, while individuals with de novo dominant toxic variants present with ID, ataxia, and cerebellar atrophy. Our combined genetic and functional data highlight the complex mutational landscape at the human RORA locus and suggest that dual mutational effects likely determine phenotypic outcome.

De novo mutations in CSNK2A1 are associated with neurodevelopmental abnormalities and dysmorphic features.

Whole exome sequencing (WES) can be used to efficiently identify de novo genetic variants associated with genetically heterogeneous conditions including intellectual disabilities. We have performed WES for 4102 (1847 female; 2255 male) intellectual disability/developmental delay cases and we report five patients with a neurodevelopmental disorder associated with developmental delay, intellectual disability, behavioral problems, hypotonia, speech problems, microcephaly, pachygyria and dysmorphic features in whom we have identified de novo missense and canonical splice site mutations in CSNK2A1, the gene encoding CK2α, the catalytic subunit of protein kinase CK2, a ubiquitous serine/threonine kinase composed of two regulatory (ß) and two catalytic (α and/or α') subunits. Somatic mutations in CSNK2A1 have been implicated in various cancers; however, this is the first study to describe a human condition associated with germline mutations in any of the CK2 subunits.

Gain-of-Function Mutations in RARB Cause Intellectual Disability with Progressive Motor Impairment.

Retinoic acid (RA) signaling plays a key role in the development and function of several systems in mammals. We previously discovered that the de novo mutations c.1159C>T (p.Arg387Cys) and c.1159C>A (p.Arg387Ser) in the RA Receptor Beta (RARB) gene cause microphthalmia and diaphragmatic hernia. However, the natural history of affected subjects beyond the prenatal or neonatal period was unknown. Here, we describe nine additional subjects with microphthalmia who have de novo mutations in RARB, including the previously described p.Arg387Cys as well as the novel c.887G>C (p.Gly296Ala) and c.638T>C (p.Leu213Pro). Moreover, we review the information on four previously reported cases. All subjects who survived the neonatal period (n = 10) displayed severe global developmental delay with progressive motor impairment due to spasticity and/or dystonia (with or without chorea). The majority of subjects also showed Chiari type I malformation and severe feeding difficulties. We previously found that p.Arg387Cys and p.Arg387Ser induce a gain-of-function. We show here that the p.Gly296Ala and p.Leu213Pro RARB mutations further promote the RA ligand-induced transcriptional activity by twofold to threefold over the wild-type receptor, also indicating a gain-of-function mechanism. These observations suggest that precise regulation of RA signaling is required for brain development and/or function in humans.

Serum response factor: positive and negative regulation of an epithelial gene expression network in the destrin mutant cornea.

Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstn(corn1) mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstn(corn1) mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstn(corn1) cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstn(corn1) cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstn(corn1) mice. Comparison of gene expression values from WT, Dstn(corn1) mutant, and Dstn(corn1) rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstn(corn1) mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstn(corn1) mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstn(corn1) mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis.

Genetic modification of corneal neovascularization in Dstn (corn1) mice.

Mutations in the gene for destrin (Dstn), an actin depolymerizing factor, lead to corneal abnormalities in mice. A null mutation in Dstn, termed Dstn (corn1) , isolated and maintained in the A.BY background (A.BY Dstn (corn1) ), results in corneal epithelial hyperproliferation, inflammation, and neovascularization. We previously reported that neovascularization in the cornea of Dstn (corn1) mice on the C57BL/6 background (B6.A.BY-Dstn (corn1) ) is significantly reduced when compared to A.BY Dstn (corn1) mice, suggesting the existence of genetic modifier(s). The purpose of this study is to identify the genetic basis of the difference in corneal neovascularization between A.BY Dstn (corn1) and B6.A.BY-Dstn (corn1) mice. We generated N2 mice for a whole-genome scan by backcrossing F1 progeny (A.BY Dstn (corn1) × B6.A.BY-Dstn (corn1) ) to B6.A.BY-Dstn (corn1) mice. N2 progeny were quantitatively phenotyped for the extent of corneal neovascularization and genotyped for markers across the mouse genome. We identified significant association of variability in corneal neovascularization with a locus on chromosome 3 (Chr3). The validity of the identified quantitative trait locus (QTL) was tested using B6 consomic mice carrying Chr3 from A/J mice. Dstn (corn1) mice from F1 and F2 intercrosses (B6.A.BY-Dstn (corn1)  × C57BL/6J-Chr3(A/J)/NaJ) were phenotyped for the extent of corneal neovascularization. This analysis showed that mice carrying the A/J allele at the QTL show significantly increased neovascularization. Our results indicate the existence of a modifier that genetically interacts with the Dstn gene. This modifier demonstrates allelic differences between C57BL6 and A.BY or A/J. The modifier is sufficient to increase neovascularization in Dstn (corn1) mice.

Differences in corneal phenotypes between destrin mutants are due to allelic difference and modified by genetic background.

PURPOSE: Mutations in destrin (Dstn) cause corneal abnormalities in mice. A null mutation, Dstn(corn1), results in corneal epithelial hyperproliferation, inflammation, and neovascularization in the A.BY background (A.BY Dstn(corn1)). Homozygosity for a point mutation, Dstn(corn1-2J), results in mild thickening of the corneal epithelium but no corneal neovascularization in a C57BL/6 (B6) background (B6 Dstn(corn1-2J)). The goal of this study was to determine whether phenotypic differences are due to allelic differences between Dstn(corn1) and Dstn(corn1-2J), or are the result of genetic background effects. METHODS: We generated two congenic (Cg) mouse lines, B6.Cg-Dstn(corn1) and A.BY.Cg-Dstn(corn1-2J), to compare to the original A.BY Dstn(corn1) and B6 Dstn(corn1-2J) lines. We performed immunohistochemistry to assay F-actin accumulation, neovascularization, proliferation, and inflammation. By western blot analysis we tested the expression of serum response factor (SRF), a known regulator of the Dstn(corn1) phenotype. RESULTS: The Dstn(corn1) mutation leads to neovascularization, hyperproliferation, and inflammation in the cornea of A.BY Dstn(corn1) as well as B6.Cg-Dstn(corn1) mice. We did not observe significant corneal neovascularization or hyperproliferation in either A.BY.Cg-Dstn(corn1-2J) or B6 Dstn(corn1-2J) mice. Actin accumulation, neovascularization, epithelial proliferation and inflammation in B6.Cg-Dstn(corn1) cornea are significantly reduced when compared to A.BY Dstn(corn1)cornea. SRF changes are consistent in Dstn(corn1) mutants, regardless of genetic background. CONCLUSIONS: Differences in the abnormal phenotypes of Dstn mutants result from allelic differences between Dstn(corn1) and Dstn(corn1-2J) . Moreover, phenotypes of Dstn(corn1) mice are modified by genetic background, suggesting the existence of genetic modifiers. Protein analysis suggests that a genetic modifier affects phenotypic severity functionally downstream from or in a pathway independent from SRF. These data demonstrate that natural genetic variation affects phenotypic severity in Dstn(corn1) mice.