Characterization of bipolar disorder patient-specific induced pluripotent stem cells from a family reveals neurodevelopmental and mRNA expression abnormalities.

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD, we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation, we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including WNT pathway components and ion channel subunits. Treatment of the CXCR4(+) NPCs with a pharmacological inhibitor of glycogen synthase kinase 3, a known regulator of WNT signaling, was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.

Retrovirus-induced insertional mutagenesis: mechanism of collagen mutation in Mov13 mice.

The Mov13 mouse strain carries a mutation in the alpha 1(I) procollagen gene which is due to the insertion of a Moloney murine leukemia provirus into the first intron. This insertion results in the de novo methylation of the provirus and flanking DNA, the alteration of chromatin structure, and the transcriptional inactivity of the collagen promoter. To address the mechanism of mutagenesis, we reintroduced a cloned and therefore demethylated version of the Mov13 mutant allele into mouse fibroblasts. The transfected gene was not transcribed, indicating that the transcriptional defect was not due to the hypermethylation. Rather, this result strongly suggests that the mutation is due to the displacement or disruption of cis-acting regulatory DNA sequences within the first intron. We also constructed a Mov13 variant allele containing a single long terminal repeat instead of the whole provirus. This construct also failed to express mRNA, indicating that the Mov13 mutation does not revert by provirus excision as has been observed for other retrovirus-induced mutations.

Analysis of exome sequence in 604 trios for recessive genotypes in schizophrenia.

Genetic associations involving both rare and common alleles have been reported for schizophrenia but there have been no systematic scans for rare recessive genotypes using fully phased trio data. Here, we use exome sequencing in 604 schizophrenia proband-parent trios to investigate the role of recessive (homozygous or compound heterozygous) nonsynonymous genotypes in the disorder. The burden of recessive genotypes was not significantly increased in probands at either a genome-wide level or in any individual gene after adjustment for multiple testing. At a system level, probands had an excess of nonsynonymous compound heterozygous genotypes (minor allele frequency, MAF ⩽ 1%) in voltage-gated sodium channels (VGSCs; eight in probands and none in parents, P = 1.5 × 10(-)(4)). Previous findings of multiple de novo loss-of-function mutations in this gene family, particularly SCN2A, in autism and intellectual disability provide biological and genetic plausibility for this finding. Pointing further to the involvement of VGSCs in schizophrenia, we found that these genes were enriched for nonsynonymous mutations (MAF ⩽ 0.1%) in cases genotyped using an exome array, (5585 schizophrenia cases and 8103 controls), and that in the trios data, synaptic proteins interacting with VGSCs were also enriched for both compound heterozygosity (P = 0.018) and de novo mutations (P = 0.04). However, we were unable to replicate the specific association with compound heterozygosity at VGSCs in an independent sample of Taiwanese schizophrenia trios (N = 614). We conclude that recessive genotypes do not appear to make a substantial contribution to schizophrenia at a genome-wide level. Although multiple lines of evidence, including several from this study, suggest that rare mutations in VGSCs contribute to the disorder, in the absence of replication of the original findings regarding compound heterozygosity, this conclusion requires evaluation in a larger sample of trios.

Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection.

To determine the requirements for the individual Ad2 E1B proteins during the transformation of rodent cells, viral mutants were constructed with genetic lesions disrupting the coding sequence of either the 175 amino acid residue (175R) or the 495 amino acid residue (495R) E1B proteins. Point mutations generating stop codons very early in the coding sequences were constructed to prevent the expression of amino-terminal protein fragments which might have biological activity. Mutant virus pm1722 contains a point mutation that terminates translation of the 175R protein after three amino acids. It was completely defective for transformation of CREF cells in virion- and DNA-mediated assays. In HeLa cells, pm1722 replicated as well as wild-type virus but produced an extreme cytopathic effect and fragmentation of host-cell DNA. Nonetheless, we provide evidence that the observed transformation defect is not due to the death of transformed cells. The mutant virus dl1520, a double mutant unable to synthesize the 495R protein, was also extremely defective for the transformation of CREF cells in virion- and viral DNA-mediated assays. This result is in contrast to studies with other Ad5 mutants with lesions in the equivalent protein. Possible explanations for this difference are discussed. Replication of dl1520 in HeLa cells was significantly reduced compared to wild-type. Studies with a third mutant virus, pm2022, which contains a stop codon after the second codon of the 495R protein, suggest that very low levels of 495R protein activity are sufficient for a productive infection and significant transforming activity.

Pumilio is essential for function but not for distribution of the Drosophila abdominal determinant Nanos.

The Drosophila gene pumilio is expressed maternally, and its function is essential during early embryogenesis for the formation of abdominal segments. Our molecular analysis reveals that pumilio is a large gene that encodes a protein of 160 kD whose RNA is enriched at the posterior pole of the egg. As with pumilio, the maternal effect gene nanos is specifically required for abdomen formation. The Nanos protein is expressed in a posterior-to-anterior concentration gradient in the developing embryo. Previous experiments demonstrated a genetic interaction between pumilio and nanos, and led to the suggestion that pumilio is required for the proper spatial distribution of the Nanos protein. Here, we show that the expression and distribution of nanos RNA and protein in embryos derived from pumilio mutant females are indistinguishable from wild type. We conclude that abdomen formation depends both on Nanos activity, spreading from the localized posterior source, and on Pumilio activity, present throughout the embryo.