Functional analysis of rare variants found in schizophrenia implicates a critical role for GIT1-PAK3 signaling in neuroplasticity.

Although the pathogenesis of schizophrenia (SCZ) is proposed to involve alterations of neural circuits via synaptic dysfunction, the underlying molecular mechanisms remain poorly understood. Recent exome sequencing studies of SCZ have uncovered numerous single-nucleotide variants (SNVs); however, the majority of these SNVs have unknown functional consequences, leaving their disease relevance uncertain. Filling this knowledge gap requires systematic application of quantitative and scalable assays to assess known and novel biological functions of genes. Here we demonstrate loss-of-function effects of multiple rare coding SNVs found in SCZ subjects in the GIT1 (G protein-coupled receptor kinase interacting ArfGAP 1) gene using functional cell-based assays involving coexpression of GIT1 and PAK3 (p21 protein (Cdc42/Rac)-activated kinase 3). Most notably, a GIT1-R283W variant reported in four independent SCZ cases was defective in activating PAK3 as well as MAPK (mitogen-activated protein kinase). Similar functional deficits were found for a de novo SCZ variant GIT1-S601N. Additional assays revealed deficits in the capacity of GIT1-R283W to stimulate PAK phosphorylation in cultured hippocampal neurons. In addition, GIT1-R283W showed deficits in the induction of GAD1 (glutamate decarboxylase 1) protein expression. Extending these functional assays to 10 additional rare GIT1 variants revealed the existence of an allelic series with the majority of the SCZ case variants exhibiting loss of function toward MAPK activation in a manner correlated with loss of PAK3 activation. Taken together, we propose that rare variants in GIT1, along with other genetic and environmental factors, cause dysregulation of PAK3 leading to synaptic deficits in SCZ.

Genome-wide association study identifies SESTD1 as a novel risk gene for lithium-responsive bipolar disorder.

This corrects the article DOI: 10.1038/mp.2015.165.

Genome-wide association study identifies SESTD1 as a novel risk gene for lithium-responsive bipolar disorder.

Lithium is the mainstay prophylactic treatment for bipolar disorder (BD), but treatment response varies considerably across individuals. Patients who respond well to lithium treatment might represent a relatively homogeneous subtype of this genetically and phenotypically diverse disorder. Here, we performed genome-wide association studies (GWAS) to identify (i) specific genetic variations influencing lithium response and (ii) genetic variants associated with risk for lithium-responsive BD. Patients with BD and controls were recruited from Sweden and the United Kingdom. GWAS were performed on 2698 patients with subjectively defined (self-reported) lithium response and 1176 patients with objectively defined (clinically documented) lithium response. We next conducted GWAS comparing lithium responders with healthy controls (1639 subjective responders and 8899 controls; 323 objective responders and 6684 controls). Meta-analyses of Swedish and UK results revealed no significant associations with lithium response within the bipolar subjects. However, when comparing lithium-responsive patients with controls, two imputed markers attained genome-wide significant associations, among which one was validated in confirmatory genotyping (rs116323614, P=2.74 × 10(-8)). It is an intronic single-nucleotide polymorphism (SNP) on chromosome 2q31.2 in the gene SEC14 and spectrin domains 1 (SESTD1), which encodes a protein involved in regulation of phospholipids. Phospholipids have been strongly implicated as lithium treatment targets. Furthermore, we estimated the proportion of variance for lithium-responsive BD explained by common variants ('SNP heritability') as 0.25 and 0.29 using two definitions of lithium response. Our results revealed a genetic variant in SESTD1 associated with risk for lithium-responsive BD, suggesting that the understanding of BD etiology could be furthered by focusing on this subtype of BD.

Copy number variation in schizophrenia in Sweden.

Schizophrenia (SCZ) is a highly heritable neuropsychiatric disorder of complex genetic etiology. Previous genome-wide surveys have revealed a greater burden of large, rare copy number variations (CNVs) in SCZ cases and identified multiple rare recurrent CNVs that increase risk of SCZ although with incomplete penetrance and pleiotropic effects. Identification of additional recurrent CNVs and biological pathways enriched for SCZ CNVs requires greater sample sizes. We conducted a genome-wide survey for CNVs associated with SCZ using a Swedish national sample (4719 cases and 5917 controls). High-confidence CNV calls were generated using genotyping array intensity data, and their effect on risk of SCZ was measured. Our data confirm increased burden of large, rare CNVs in SCZ cases as well as significant associations for recurrent 16p11.2 duplications, 22q11.2 deletions and 3q29 deletions. We report a novel association for 17q12 duplications (odds ratio=4.16, P=0.018), previously associated with autism and mental retardation but not SCZ. Intriguingly, gene set association analyses implicate biological pathways previously associated with SCZ through common variation and exome sequencing (calcium channel signaling and binding partners of the fragile X mental retardation protein). We found significantly increased burden of the largest CNVs (>500 kb) in genes present in the postsynaptic density, in genomic regions implicated via SCZ genome-wide association studies and in gene products localized to mitochondria and cytoplasm. Our findings suggest that multiple lines of genomic inquiry--genome-wide screens for CNVs, common variation and exonic variation--are converging on similar sets of pathways and/or genes.

Genome-wide association study in a Swedish population yields support for greater CNV and MHC involvement in schizophrenia compared with bipolar disorder.

Schizophrenia (SCZ) and bipolar disorder (BD) are highly heritable psychiatric disorders with overlapping susceptibility loci and symptomatology. We conducted a genome-wide association study (GWAS) of these disorders in a large Swedish sample. We report a new and independent case-control analysis of 1507 SCZ cases, 836 BD cases and 2093 controls. No single-nucleotide polymorphisms (SNPs) achieved significance in these new samples; however, combining new and previously reported SCZ samples (2111 SCZ and 2535 controls) revealed a genome-wide significant association in the major histocompatibility complex (MHC) region (rs886424, P=4.54 × 10(-8)). Imputation using multiple reference panels and meta-analysis with the Psychiatric Genomics Consortium SCZ results underscored the broad, significant association in the MHC region in the full SCZ sample. We evaluated the role of copy number variants (CNVs) in these subjects. As in prior reports, deletions were enriched in SCZ, but not BD cases compared with controls. Singleton deletions were more frequent in both case groups compared with controls (SCZ: P=0.003, BD: P=0.013), whereas the largest CNVs (>500 kb) were significantly enriched only in SCZ cases (P=0.0035). Two CNVs with previously reported SCZ associations were also overrepresented in this SCZ sample: 16p11.2 duplications (P=0.0035) and 22q11 deletions (P=0.03). These results reinforce prior reports of significant MHC and CNV associations in SCZ, but not BD.

Growth of factor-dependent hemopoietic precursor cell lines.

Cell lines have been produced from long-term cultures of mouse bone marrow that require a factor, present in WEHI-3 conditioned medium (CM) or in spleen CM, for their sustained growth. The cell lines were obtained from nonvirus-treated cultures, are nonleukemic, maintain a normal karyotype, and form colonies showing granulocyte maturation when plated in soft agar. Granulocyte/macrophage (GM) colony-stimulating factor is not the inductive moiety involved in the maintenance of proliferation of these cells. It is suggested that the cell lines represent a self-renewing population of cells ancestral to GM colony-forming cells, which may be responding to a hitherto unrecognized regulator.

Friend disease in vitro.

In long-term marrow cultures, hemopoiesis can be maintained for several months, although erythropoiesis is normally suppressed at the most primitive level of development (the erythroid colony-forming cells). Infection of these cultures with a viral complex combining helper-independent murine leukemia virus (F-MuLV) and a spleen focus-forming virus (SFFVp) results in a productive infection of both the replication defective SFFVp and the F-MuLV. After infection, the cultures show a dramatic elevation in the numbers of late erythroid progenitor cells (CFU-E), many of which will grow in the absence of added erythropoietin, and a transient erythropoietin, independent erythropoiesis, including the production of mature, enucleated erythrocytes. Hemopoiesis eventually declines, with no evidence for the generation of Friend tumor cells. When erythropoiesis is induced in the long-term cultures by addition of anemic mouse serum before infection by polycythemia-inducing Friend virus, the generation of erythropoietin-independent CFU-E and erythrocyte formation is followed by the sustained production (greater than 40 wk) of primitive erythroid cells with low spontaneous levels (less than 5%) of hemoglobinization. Although these cells will produce spleen colonies in irradiated mice and can be cloned in soft-gel media, they do not produce autonomous, permanently growing cell lines in vitro, i.e., they retain a dependency upon the marrow-adherent layer for their continued growth. However, following a further passage on a "virgin" marrow environment, permanent cell lines can be established that are able to grow independently of environmental influences. Thus, this system is the first description of a complete in vitro system for the reproducible production and isolation of Friend virus-induced erythroid cell lines.

Type-specific radioimmunoassays for the gp70s of mink cell focus-inducing murine leukemia viruses: expression of a cross-reacting antigen in cells infected with the Friend strain of the spleen focus-forming virus.

We have isolated the gp70 of a helper-independent strain of a Friend mink cell focus-inducing (MCF) virus, Fr-MCF-1. This recombinant virus, like the previously described AKR-MCF viruses, has been shown by both biological and biochemical means to be an envelope gene recombinant between Friend murine leukemia virus (F-MuLV) and a mouse xenotropic virus. Utilizing (125)I- labeled Fr-MCF-1 gp70 and antiserum prepared against an MCF strain of Moloney type-C virus (Mol-MCF(83)), we have developed a radioimmunoassay which detects immunological determinant (s)contained in the gp70s of MCF viruses derived from F-MuLV, Mol-MuLV, and AKR-MuLV. This MCF determinant(s) is not detected in the ecotropic parents of each of these MCF viruses, nor in helper-independent murine xenotropic viruses derived from Swiss or BALB/c mice. A protein partially cross-reactive with the MCF gp70 determinant(s) is detected in a replicating xenotropic virus derived from NZB mice. Utilizing this MCF gp70 specific immunoassay, we can detect a cross-reacting gene product coded for by the Friend strain of the spleen focus-forming virus (SFFV) in rat fibroblasts nonproductively infected with SFFV. The results support earlier molecular hybridization studies which indicated that the genome of SFFV contains genetic information derived from both F-MuLV and xenotropic virus, and that the xenotropic-related sequences in SFFV are highly related to those found in MCF murine type-C viruses.

Radioimmunoassay of mammalian type C viral proteins. 3. Detection of viral antigen in normal murine cells and tissues.

A radioimmunoassay specific for a murine leukemia virus structural protein, the gs antigen, detects an antigenic reactivity in normal murine cells in culture and natural tissues. The assay was shown to measure an antigen that is highly related to the virion protein as shown by absorption tests, immunoadsorbent chromatography, and by analysis of linearized dose-response curves. These findings combined with the finding of viral-specific RNA indicate that portions of the viral genome are being expressed with a much greater frequency than previously appreciated.

Helper-independent mink cell focus-inducing strains of Friend murine type-C virus: potential relationship to the origin of replication-defective spleen focus-forming virus.

Recent studies have indicated that both the replication-defective spleen focus-forming virus (SFFV) in the Friend virus complex and the helper-independent mink cell focus-inducing (MCF) viruses derived from AKR-murine leukemia virus (MuLV) are env gene recombinants between ecotropic virus and xenotropic virus. In an attempt to isolate additional env gene recombinants between Friend murine leukemia virus (F-MuLV) and xenotropic virus, we have inoculated cloned ecotropic F-MuLV into newborn NIH Swiss mice and analyzed MuLV released from preleukemic and leukemic spleens of infected mice. Two helper-independent MCF strains of F-MuLV have been isolated. Like the previously described AKR-MCF viruses, the Friend MCF viruses are env gene recombinants between an ecotropic virus (F-MuLV) and a mouse xenotropic virus, as shown by host range, interference pattern, and tryptic peptide analysis of the gp70s of these MuLV. Furthermore, RNA from the Friend MCF viruses hybridizes completely to cDNAsffv, a nucleic acid probe which detects that portion of SFFV which was not derived from P-MuLV. The ability to isolate replicating MCF viruses derived from F-MuLV FURTHER strengthens the parallels between the Friend erythroleukemia system and the AKR thymic leukemia system. Finally, the potential relationship of helper-independent env gene recombinants between F-MuLV and xenotropic virus to be highly leukemogenic SFFV is discussed.

Whole-genome association study of bipolar disorder.

We performed a genome-wide association scan in 1461 patients with bipolar (BP) 1 disorder, 2008 controls drawn from the Systematic Treatment Enhancement Program for Bipolar Disorder and the University College London sample collections with successful genotyping for 372,193 single nucleotide polymorphisms (SNPs). Our strongest single SNP results are found in myosin5B (MYO5B; P=1.66 x 10(-7)) and tetraspanin-8 (TSPAN8; P=6.11 x 10(-7)). Haplotype analysis further supported single SNP results highlighting MYO5B, TSPAN8 and the epidermal growth factor receptor (MYO5B; P=2.04 x 10(-8), TSPAN8; P=7.57 x 10(-7) and EGFR; P=8.36 x 10(-8)). For replication, we genotyped 304 SNPs in family-based NIMH samples (n=409 trios) and University of Edinburgh case-control samples (n=365 cases, 351 controls) that did not provide independent replication after correction for multiple testing. A comparison of our strongest associations with the genome-wide scan of 1868 patients with BP disorder and 2938 controls who completed the scan as part of the Wellcome Trust Case-Control Consortium indicates concordant signals for SNPs within the voltage-dependent calcium channel, L-type, alpha 1C subunit (CACNA1C) gene. Given the heritability of BP disorder, the lack of agreement between studies emphasizes that susceptibility alleles are likely to be modest in effect size and require even larger samples for detection.

Dexamethasone stimulation of murine mammary tumor virus expression: a tissue culture source of virus.

In mouse cell lines derived from mammary adenocarcinomnas, the synthetic steroid dexamnethasone stimulates production of murine mammary tumor virus. Viral RNA and antigens are increased as much as 20-fold, and culture fluid supernatants from steroid-treated cells contain type B particles with reverse transcriptase. These cells provide a possible tissue culture source of this virus and a model system for studying the mechanism of action of corticosteroids and the regulation of transcription of integrated viral DNA.

Induction of murine C-type viruses from clonal lines of virus-free BALB-3T3 cells.

Each clone of BALB/c mouse embryo cells that has been tested can be induced to form C-type virus. The individual cells therefore contain a complete copy of the genetic information for making the murine RNA tumor viruses.

Reverse transcriptases of primate viruses as immunological markers.

Antibodies were prepared against the DNA polymerases (reverse transcriptases) of three potentially oncogenic RNA viruses of primates. Two type C viruses, isolated from a woolly monkey fibrosarcoma and from a gibbon ape lymphosarcoma, have polymerases that are immunologically related to each other and are distinct from the type C viruses isolated from other mammals.

Increased sarcoma virus RNA in cells transformed by leukemia viruses: model for leukemogenesis.

A morphologically flat revertant of mink cells nonproductively infected with Moloney sarcoma virus exhibited contact inhibition and lacked detectable sarcoma virus RNA. Superinfection by usually nontransforming type C mammalian leukemia-causing viruses induced transformation and increased sarcoma virus RNA. The results suggest a model for leukemogenesis in animals by increasing, during replication of usually nontransforming leukemia viruses, the levels of RNA from potentially oncogenic cell or integrated virus transforming genes.

Transformation by cloned Harvey murine sarcoma virus DNA: efficiency increased by long terminal repeat DNA.

The coding sequences for the transforming (src) protein (p21) of Harvey murine sarcoma virus have been localized to a 1.3 kilobase pair segment near the 5' end of the viral genome. Ligation of the viral terminal repeat DNA to the left end of the src region DNA markedly enhanced the low transforming efficiency of the src region DNA.

Sequential translation of trinucleotide codons for the initiation and termination of protein synthesis.

Terminal events in protein synthesis were studied with trinucleotide codons. Initiator and terminator trinucleotides sequentially stimulate N-formyl-methionyl-tRNA binding to ribosomes and the release of free N-formyl-methionine from the ribosomal intermediate. The release factor discovered by Capecchi is also required. The trinucleotides UGA, UAA, and UAG were found to be terminator codons. This pattern of codon degeneracy has not been observed with other trinucleotides and transfer RNA.

Mammalian and yeast ras gene products: biological function in their heterologous systems.

Activated versions of ras genes have been found in various types of malignant tumors. The normal versions of these genes are found in organisms as diverse as mammals and yeasts. Yeast cells that lack their functional ras genes, RASSC-1 and RASSC-2, are ordinarily nonviable. They have now been shown to remain viable if they carry a mammalian rasH gene. In addition, yeast-mammalian hybrid genes and a deletion mutant yeast RASSC-1 gene were shown to induce morphologic transformation of mouse NIH 3T3 cells when the genes had a point mutation analogous to one that increases the transforming activity of mammalian ras genes. The results establish the functional relevance of the yeast system to the genetics and biochemistry of cellular transformation induced by mammalian ras genes.

Nucleotide sequence of the p21 transforming protein of Harvey murine sarcoma virus.

Harvey murine sarcoma virus is a retrovirus which transforms cells by means of a single virally encoded protein called p21 has. We have determined the nucleotide sequence of 1.0 kilobase in the 5' half of the viral genome which encompasses the has coding sequences and its associated regulatory signals. The nucleotide sequence has identified the amino acid sequence of two additional overlapping polypeptides which share their reading frames and the carboxyl termini with p21 but which contain additional NH2-terminal amino acids.

Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor.

To acquire transforming potential, the precursor of the Ras oncoprotein must undergo farnesylation of the cysteine residue located in a carboxyl-terminal tetrapeptide. Inhibitors of the enzyme that catalyzes this modification, farnesyl protein transferase (FPTase), have therefore been suggested as anticancer agents for tumors in which Ras contributes to transformation. The tetrapeptide analog L-731,735 is a potent and selective inhibitor of FPTase in vitro. A prodrug of this compound, L-731,734, inhibited Ras processing in cells transformed with v-ras. L-731,734 decreased the ability of v-ras-transformed cells to form colonies in soft agar but had no effect on the efficiency of colony formation of cells transformed by either the v-raf or v-mos oncogenes. The results demonstrate selective inhibition of ras-dependent cell transformation with a synthetic organic inhibitor of FPTase.