De novo variants predicting haploinsufficiency for DIP2C are associated with expressive speech delay.

The disconnected (disco)-interacting protein 2 (DIP2) gene was first identified in D. melanogaster and contains a DNA methyltransferase-associated protein 1 (DMAP1) binding domain, Acyl-CoA synthetase domain and AMP-binding sites. DIP2 regulates axonal bifurcation of the mushroom body neurons in D. melanogaster and is required for axonal regeneration in the neurons of C. elegans. The DIP2 homologues in vertebrates, Disco-interacting protein 2 homolog A (DIP2A), Disco-interacting protein 2 homolog B (DIP2B), and Disco-interacting protein 2 homolog C (DIP2C), are highly conserved and expressed widely in the central nervous system. Although there is evidence that DIP2C plays a role in cognition, reports of pathogenic variants in these genes are rare and their significance is uncertain. We present 23 individuals with heterozygous DIP2C variants, all manifesting developmental delays that primarily affect expressive language and speech articulation. Eight patients had de novo variants predicting loss-of-function in the DIP2C gene, two patients had de novo missense variants, three had paternally inherited loss of function variants and six had maternally inherited loss-of-function variants, while inheritance was unknown for four variants. Four patients had cardiac defects (hypertrophic cardiomyopathy, atrial septal defects, and bicuspid aortic valve). Minor facial anomalies were inconsistent but included a high anterior hairline with a long forehead, broad nasal tip, and ear anomalies. Brainspan analysis showed elevated DIP2C expression in the human neocortex at 10-24 weeks after conception. With the cases presented herein, we provide phenotypic and genotypic data supporting the association between loss-of-function variants in DIP2C with a neurocognitive phenotype.

Novel DIP2C gene splicing variant in an individual with focal infantile epilepsy.

Disco-interacting protein 2 C (DIP2C) encodes a disco-interacting protein and is highly expressed in the nervous system. Most variants of DIP2C are microdeletions on chromosome 10p15.3. This study reports a 17-month-old infant with focal infantile epilepsy who has a single-nucleotide variation in DIP2C that results in alternative splicing. The de novo variation (NM_014974.3: c.1057+2T>G) in DIP2C was uncovered through whole-exome sequencing. Minigene assays were performed and verified the alternative splicing caused by the variation. Finally, an 80-bp nucleotide deletion in the 3' end of Exon 8 was detected. Our study identified a de novo splicing variant that affects the coding length of DIP2C. This finding provides a new candidate gene for focal infantile epilepsy. Importantly, our finding is the first to associate a single nucleotide variant in DIP2C with focal infantile epilepsy.

Brain transcriptome study through CRISPR/Cas9 mediated mouse Dip2c gene knock-out.

Dip2C is highly expressed in brain and many other tissues but its biological functions are still not clear. Genes regulated by Dip2C in brain have never been studied. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, adaptive immune systems of bacteria and archaea, have been recently developed and broadly used in genome editing. Here, we describe targeted gene deletions of Dip2c gene in mice via CRISPR/Cas9 system and study of brain transcriptome under Dip2C regulation. The CRISPR/Cas9 system effectively generated targeted deletions of Dip2c by pronuclei injection of plasmids that express Cas9 protein and two sgRNAs. We achieved targeted large fragment deletion with efficiencies at 14.3% (1/7), 66.7% (2/3) and 20% (1/5) respectively in 3 independent experiments, averaging 26.7%. The large deletion DNA segments are 160.4 kb (Dip2CΔ160kb), spanning from end of exon 4 to mid of exon 38. A mouse with two base pair deletion was generated from a single sgRNA targeting in exon 4 (Dip2cΔ2bp) by non-homologous end joining (NHEJ). Loss of gene expression for Dip2c mRNA was confirmed by quantitative real-time PCR (qPCR). Dip2C-regulated genes and pathways in brain were investigated through RNAseq of Dip2cΔ2bp. In total, 838 genes were found differentially regulated, with 252 up and 586 down. Gene ontology (GO) analysis indicated that DEGs in brain are enriched in neurological functions including 'memory', 'neuropeptide signaling pathway', and 'response to amphetamine' while KEGG analysis shows that 'neuroactive ligand-receptor interaction pathway' is the most significantly enriched. DEGs Grid2ip, Grin2a, Grin2c, Grm4, Gabbr2, Gabra5, Gabre, Gabrq, Gabra6 and Gabrr2 are among the highly regulated genes by Dip2C. Results confirm Dip2C may play important roles in brain development and function.

DIP2C expression in breast cancer and its clinical significance.

INTRODUCTION: The aim of this study was to investigate DIP2C expression in different subtypes of breast cancer tissues and cell lines and its correlation with clinicopathologic and histopathological features, in an effort to elucidate the DIP2C expression profile in breast cancer and its clinical significance. METHODS: Hereby, we investigated the DIP2C expression in breast cancer tissues using TMA-IHC method and the DIP2C expression in breast cell lines using quantitative RT-PCR. RESULTS: DIP2C displayed universal expression, being present in all the breast cancer subtypes. There were more cases that staining weakly in breast cancer tissues (n=79/150, 52.7%) than that in fibroadenomas tissues (n=2/18, 11.1%) and normal tissues (n=2/20, 10.0%) (χ2=21.84, P <0.001). Within different intrinsic subtypes of breast cancer assayed by IHC expression profiles, there were less cases of the strongly staining group in basal-like subtype (n=38/86, 44.2%) and HER-2 subtype (n=6/24, 25.0%) than that in luminal A (14/20, 70%) and luminal B (13/20, 65%) subtypes (χ2=11.77, p=0.008). Furthermore, DIP2C expression was positive correlated with ER (χ2=8.90, p=0.003) and PR expression (χ2=10.94, p=0.001), while negative correlated with EGFR expression (χ2=9.27, p=0.002), in accordance with the results of cell lines with different subtypes. Oncomine database also confirmed that, DIP2C was expressed lower in breast cancer tissues, and could indicate prognosis. CONCLUSION: our data revealed DIP2C expression level decreased in breast cancer, especially in basal-like and HER-2 subtypes, and could be a valuable target for diagnosis on specific subtype of breast cancer.

Loss of DIP2C in RKO cells stimulates changes in DNA methylation and epithelial-mesenchymal transition.

BACKGROUND: The disco-interacting protein 2 homolog C (DIP2C) gene is an uncharacterized gene found mutated in a subset of breast and lung cancers. To understand the role of DIP2C in tumour development we studied the gene in human cancer cells. METHODS: We engineered human DIP2C knockout cells by genome editing in cancer cells. The growth properties of the engineered cells were characterised and transcriptome and methylation analyses were carried out to identify pathways deregulated by inactivation of DIP2C. Effects on cell death pathways and epithelial-mesenchymal transition traits were studied based on the results from expression profiling. RESULTS: Knockout of DIP2C in RKO cells resulted in cell enlargement and growth retardation. Expression profiling revealed 780 genes for which the expression level was affected by the loss of DIP2C, including the tumour-suppressor encoding CDKN2A gene, the epithelial-mesenchymal transition (EMT) regulator-encoding ZEB1, and CD44 and CD24 that encode breast cancer stem cell markers. Analysis of DNA methylation showed more than 30,000 sites affected by differential methylation, the majority of which were hypomethylated following loss of DIP2C. Changes in DNA methylation at promoter regions were strongly correlated to changes in gene expression, and genes involved with EMT and cell death were enriched among the differentially regulated genes. The DIP2C knockout cells had higher wound closing capacity and showed an increase in the proportion of cells positive for cellular senescence markers. CONCLUSIONS: Loss of DIP2C triggers substantial DNA methylation and gene expression changes, cellular senescence and epithelial-mesenchymal transition in cancer cells.