Double Mutant Gene Research Articles

PIK3CA-TP53 co-mutation as a biomarker of overall survival in malignant tumor.

e15001 Background: Overall survival (OS) is an important endpoint, with the advantage that there is minimal ambiguity in defining an OS event; the patient is either alive or dead. Some single gene mutations, such as TP53, were identified as important predictors of shorter OS in malignant tumors. However, the relationship between gene co-mutation and OS of malignant tumors is poorly defined. Methods: Genomic and OS data were derived from The Cancer Genome Atlas (TCGA) databases. We defined the double-mutant gene pair as “hit pair” in the specific cancer type if statistically significant OS difference was observed in at least one of three comparison (double-mutant samples vs gene A-only-mutant samples; double-mutant samples vs gene B-only-mutant samples; gene A-only-mutant samples vs gene B-only-mutant samples). Results: PIK3CA-TP53 co-mutation was evaluated as “hit pair” in seven cancer types: GBM (Glioblastoma multiforme), HNSC (Head and neck squamous cell carcinoma), LUSC (Lung squamous cell carcinoma), SARC (Sarcoma), SKCM (Skin cutaneous melanoma), UCEC (Uterine corpus endometrial carcinoma), and UCS (Uterine carcinosarcoma). In SARC and SKCM, the expected hazard ratio was higher in both-mutant group than TP53-only-mutant group, and neither genes were correlated with OS. Especially in SKCM, after we took “neither” group (both genes were wild-type) into comparison, hazard ratio of both-mutant group was significantly higher than neither group. Single-gene mutation didn’t make difference on hazard ratio, which indicated that the both-mutant interaction made the leading contribution. In GBM, LUSC, and UCS, the significant difference of hazard ratio between TP53-only-mutant group and PIK3CA-only-mutant group was neutralized in the both-mutant group, to some extent equivalent to “average effect (OS curve of double-mutant group was between that of only single gene mutant groups)". For PIK3CA-TP53 in HNSC, both-mutant group and TP53-only-mutant group carried with a higher hazard ratio than PIK3CA-only-mutant group. It suggested that the increasing hazard ratio in both-mutant group might be resulted from TP53 mutation. For the circumstance in UCEC, TP53 and PIK3CA were testified to be correlated with higher and lower hazard ratio, respectively and both-mutant group and TP53-only-mutant group both with significantly higher hazard ratio than PIK3CA-only-mutant group, which further verified the greater strength of TP53 than PIK3CA in both-mutant group. Conclusions: In summary, our study identified PIK3CA-TP53 co-mutation as a predictor of OS in seven cancer types: GBM, HNSC, LUSC, SARC, SKCM, UCEC, and UCS, especially in SARC and SKCM.

CTNNB1-Mutant Aldosterone-Producing Adenomas With Somatic Mutations of GNA11/GNAQ Have Distinct Phenotype and Genotype

Background: We report (this meeting) somatic mutation of GNA11/Q in CTNNB1-mutant APAs. The recurrent co-driver mutation causes reversible hypertension in puberty, pregnancy, or menopause. We have investigated the molecular mechanism of this association. Methods: Gene expression profiles in 3 double mutant APAs were studied by unsupervised hierarchical clustering analysis and enrichment analysis of 362 differentially expressed genes and validated by qPCR, IFC and IHC in 10 double mutant APAs or transfected primary adrenal cells. Multiple region biopsies were performed in 7 adrenals adjacent to double-mutant APAs and 4 APAs with KCNJ5 or CACNA1D mutations. The findings of APA mutations in adjacent adrenals were replicated in each case by ddPCR ± NGS. Results: Unsupervised hierarchical clustering analysis showed clustering of the double-mutant APAs, and a high proportion of genes were many-fold upregulated compared to other APAs. LHCGR, TMEM132E, DKK1, C9orf84, FAP, GNRHR and MPP3 are among the genes with high expression. A small number of genes are down-regulated in the double-mutant APAs, including CYP11B1. qPCR confirmed an average of ~10 to 1000-fold higher expression of the hallmark genes in double-mutants. Enrichment analysis showed significant enrichment of features or terms concerned with cell junction and cell adhesion (P<10–8). IFC confirmed LHCGR intensity was 31–144 fold higher in primary adrenal cells with GNA11-p.Gln209Pro transfection and high CTNNB1 intensity. LHCGR intensity was qualitatively and quantitatively associated with immunofluorescence for CTNNB1. IHC of double-mutant APAs showed absent CYP11B1 but strong staining of CYP11B2. qPCR confirmed a lower CYP11B1/CYP11B2 ratio and a higher LHCGR expression (P<10–3, both). IHC confirmed LHCGR positivity in double-mutant APAs but distribution varied both within and between cells. Adjacent ZG was hyperplastic, with absence of both CYP11B1 and CYP11B2 staining, but weak/moderate staining for LHCGR. The same GNA11 ± CTNNB1 somatic mutations were detected in multiple regions of the adjacent adrenals to 3 double mutant APAs. qPCR of hallmark APA genes differed from the APAs. High concordance between ddPCR, NGS and Sanger sequencing of the findings of APA mutations in adjacent adrenals when analysed in the same sample. No mutations were found in 4 adrenals adjacent to APAs with KCNJ5 or CACNA1D mutations, nor in other 4 adrenals adjacent to double-mutant APAs. Conclusions: Patients harboring APAs with somatic mutations in both GNA11/GNAQ Q209 and CTNNB1 have distinct phenotype in both the APAs and their adjacent adrenals. Same GNA11 ± CTNNB1 somatic mutations were found in the adjacent adrenals to double mutant APAs. We infer that a double-hit within related pathways is more likely than a single-hit to cause large increases in expression of LHCGR, and of other genes which may influence clinical presentation.

A DNA Vaccine Encoding SA-4-1BBL Fused to HPV-16 E7 Antigen Has Prophylactic and Therapeutic Efficacy in a Cervical Cancer Mouse Model.

The SA-4-1BBL, an oligomeric novel form of the natural ligand for the 4-1BB co-stimulatory receptor of the tumor necrosis factor (TNF) superfamily, as a recombinant protein has potent pleiotropic effects on cells of innate, adaptive, and regulatory immunity with demonstrated therapeutic efficacy in several tumor models. However, the production of soluble form of SA-4-1BBL protein and quality control is time and resource intensive and face various issues pertinent to clinical development of biologics. The present study sought to take advantage of the simplicity and translatability of DNA-based vaccines for the production and delivery of SA-4-1BBL for cancer immune prevention and therapy. A chimeric HPV-16 E7 DNA vaccine (SP-SA-E7-4-1BBL) was constructed that contains the signal peptide (SP) of calreticulin (CRT), streptavidin (SA) domain of SA-4-1BBL, HPV-16 E7 double mutant gene, and the extracellular domain of mouse 4-1BBL. Immunization by gene gun with SP-SA-E7-4-1BBL induced greater prophylactic as well as therapeutic effects in C57BL/6 mice against TC-1 tumor model compared with immunization with E7wt, SP-SA-4-1BBL or reference-positive control CRT-E7wt. The therapeutic efficacy of the DNA vaccine was associated with increased frequency of E7-specific T cells producing interferon (IFN)-γ. Overall, our data suggest that this DNA-based vaccine strategy might represent a translational approach because it provides a simpler and versatile alternative to a subunit vaccine based on SA-4-1BBL and E7 proteins.

Gender differences of peripheral plasma and liver metabolic profiling in APP/PS1 transgenic AD mice

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment. Currently, there is less knowledge of the involvement of the peripheral biofluid/organ in AD, compared with the central nervous system. In addition, with reported high morbidity in women in particular, it has become very important to explore whether gender difference in the peripheral metabolome is associated with AD. Here, we investigated metabolic responses of both plasma and liver tissues using an APP/PS1 double mutant transgenic mouse model with NMR spectroscopy, as well as analysis from serum biochemistry and histological staining. Fatty acid composition from plasma and liver extracts was analyzed using GC-FID/MS. We found clear gender differences in AD transgenic mice when compared with their wild-type counterparts. Female AD mice displayed more intensive responses, which were highlighted by higher levels of lipids, 3-hydroxybutyrate and nucleotide-related metabolites, together with lower levels of glucose. These observations indicate that AD induces oxidative stress and impairs cellular energy metabolism in peripheral organs. Disturbances in AD male mice were milder with depletion of monounsaturated fatty acids. We also observed a higher activity of delta-6-desaturate and suppressed activity of delta-5-desaturate in female mice, whereas inhibited stearoyl-CoA-desaturase in male mice suggested that AD induced by the double mutant genes results in different fatty acids catabolism depending on gender. Our results provide metabolic clues into the peripheral biofluid/organs involved in AD, and we propose that a gender-specific scheme for AD treatment in men and women may be required.

Mutations in the Nuclear Encoded Novel Mitochondrial Protein CHCHD10 Cause an Autosomal Dominant Mitochondrial Myopathy (IN7-2.002)

Objective: To report a novel causative gene for an autosomal dominant mitochondrial myopathy. Background Mitochondrial myopathies belong to a larger group of systemic diseases caused by morphological or biochemical abnormalities of mitochondria. Mitochondrial disorders can be caused by mutation either in the mitochondrial or in the nuclear genome. Design/Methods: Patients are part of previously reported Hispanic kindred with autosomal dominant mitochondrial myopathy (Heiman-Patterson et al 1997). Linkage analysis was established using fluorescent microsatellite markers and illumina genome wide SNP markers. Candidate gene was identified using MitoCarta (Vamsi K. Mootha, personal communication). Candidate gene was sequenced using Beckman Coulter CEQ 8000 DNA analysis system. The cDNA of wild type, single and double mutant gene were cloned in pCMV-Myc vector and co-transfected with pDsRed2-mito in HEK293 cells. The co-localization was monitored with confocal microscopy. Results: In this five generation Hispanic family we established linkage to chromosome 22q11. Using the tools of integrative gneomics, we established CHCHD10/ C22orf16 as the high scoring mitochondrial candidate gene in our minimal candidate region. Sequence analysis revealed a double heterozygote missense mutation in this gene. These two mutations segregated with disease phenotype and were absent in 500 Caucasians and Hispanic non affected controls. Both wild types, single and double mutant proteins co-localized with mitochondria. Conclusions: We identified a new nuclear encoded mitochondrial gene ( CHCHD10 ) as the cause of an autosomal dominant mitochondrial myopathy. CHCHD10 encodes a non characterized 142 aa protein with a target mitochondrial signaling peptide and a conserved CHCH domain. This CHCH domain is present in many intermembrane space proteins, such as the family of small Tim proteins. The exact function of this protein remains to be determined. Our findings expands the spectrum of mitochondrial myopathies caused by nuclear gene dysfunction. Disclosure: Dr. Ajroud-Driss has nothing to disclose. Dr. Fecto has nothing to disclose. Dr. Ajroud has nothing to disclose. Dr. Siddique has nothing to disclose.

Central carbon metabolism influences fidelity of DNA replication in Escherichia coli

Recent studies indicated that there is a direct link between central carbon metabolism (CCM) and initiation and elongation of DNA replication in Eschericha coli. Namely, effects of certain mutations in genes coding for replication proteins (dnaA, dnaB, dnaE, dnaG, and dnaN) could be specifically suppressed by deletions of some genes, whose products are involved in CCM reactions (pta, ackA, pgi, tktB, and gpmA). Here, we demonstrate that the link between CCM and DNA synthesis can be extended to the DNA replication fidelity, as we report changes of the mutator phenotypes of E. coli dnaQ49 and dnaX36 mutants (either suppression or enhancement) by dysfunctions of zwf, pta, ackA, acnB, and icdA genes. Overexpression of appropriate wild-type CCM genes in double mutants resulted in reversions to the initial mutator phenotypes, indicating that the effects were specific. Moreover, the observed suppression and enhancement effects were not caused by changes in bacterial growth rates. These results suggest that there is a genetic correlation between CCM and DNA replication fidelity in E. coli, apart from the already documented link between CCM and DNA replication initiation control and elongation efficiency.

The specificity of cytokinin signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the receptors

Arabidopsis thaliana has three membrane-located cytokinin receptors (AHK2, AHK3 and CRE1/AHK4), which are sensor histidine kinases containing a ligand-binding CHASE domain. Despite their structural similarity the role of these receptors differs in planta. Here we have explored which parameters contribute to signal specification. In a bacterial assay, the CHASE domain of AHK2 has a similar ligand binding spectrum as CRE1/AHK4. It shows the highest affinity for isopentenyladenine (iP) and trans-zeatin (tZ) with an apparent K(D) of 1.4 and 4.0 nm, respectively. Real-time PCR analysis of cytokinin primary response genes in double mutants retaining only single receptors revealed that all receptors are activated in planta by cytokinin concentrations in the low nanomolar range. However, there are differences in sensitivity towards the principal cytokinins iP and tZ. The activation of the cytokinin-sensitive P(ARR5) :GUS reporter gene in three different double mutants shows specific, but also overlapping, spatial domains of activity, which were for all receptors predominantly in the shoot apical meristems and root cap columella. AHK2 and AHK3 signal specifically in leaf parenchyma cells, AHK3 in stomata cells, and CRE1/AHK4 in the root vasculature. Promoter-swap experiments demonstrate that CRE1/AHK4 can functionally replace AHK2 but not AHK3. However, the cytoplasmic AHK3 histidine kinase (Hk) domain can be replaced by the CRE1/AHK4 Hk domain, which suggests that functionality is mediated in this case by the extracytosolic domain. Together, the data show that both differential gene expression and ligand preference contribute to specify the receptor activity.

Genetic analysis of an essential cytoplasmic domain of Escherichia coli SecY based on resistance to Syd, a SecY-interacting protein.

We previously described a dominant negative secY-d1 allele in Escherichia coli, whose product interferes with protein export, presumably by sequestering SecE, the stabilizing partner of SecY. Syd is the product of a multicopy suppressor of the secY-d1 phenotype, and its overproduction preferentially stabilizes the wild-type SecY protein. In contrast, overproduction of Syd is toxic to the secY24 mutant, which shows a partial defect in SecY-SecE interaction. We isolated Syd-resistant revertants from the secY24 mutant. Pseudo-reversions mapped to sites at or near the secY24 mutation site (Gly240-->Asp). The secY249 mutation (Ala249-->Val) intragenically suppressed Syd sensitivity, but not the temperature-sensitive Sec phenotype of the secY24 mutation. The SecY249 mutant protein shows a reduced capacity to be stabilized by Syd, suggesting that the mutation weakens the SecY-Syd interaction. The other two mutations changed residue 240 (the site of the secY24 alteration) to Asn (secY245) or Ala (secY241) and restored the ability of the cell to export protein. Although the secY245 mutant retained some sensitivity to Syd overproduction, the secY241 mutant was completely Syd-resistant. Furthermore, the secY241 mutation seemed to represent a "hyper reversion" with respect to the SecY-SecE interaction. Protein export in this mutant was no longer sensitive to SecY-d1. When the secY-d1 mutation was combined intragenically with secY241, the resulting double mutant gene (secY-d1-241) showed an increased ability to interfere with protein export. On the basis of our model for SecY-d1, these results suggest that the secY241 alteration enhances SecY-SecE interaction. These results indicate that residue 240 of SecY is crucial for the interaction between the cytosolic domains of SecY and SecE required for the establishment of the translocase complex.

Reduced Growth Cone Motility in Cultured Neurons fromDrosophilaMemory Mutants with a Defective cAMP Cascade

Drosophila memory mutants dunce (dnc) and rutabaga (rut) are known to have altered intracellular cAMP levels, nerve terminal growth, and plasticity of synaptic transmission. Because the growth cone is responsible for neurite outgrowth and synaptogenesis, video microscopy was used to examine growth cone morphology and behavior of mutant neurons in larval CNS cultures. We found that growth cone exploratory movement was nearly arrested by both mutations, even though they change cAMP levels in opposite directions. The dnc phenotype could be mimicked by normal neurons when perfused with dibutyryl cAMP (db-cAMP) or forskolin. In contrast, rut growth cones became active when perfused with db-cAMP. Furthermore, motility was also restored by counterbalancing the effects of the two genes in double mutants, indicating that dynamic control of growth cone motility in developing Drosophila neurons requires optimal cAMP levels within an operational range. These findings represent the first demonstration of altered growth cone properties in learning and memory mutants and establish in a natural setting the role of cAMP in growth cone motility and neuronal plasticity.

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to beta-galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229-->Ile) and Val274 (Asp274-->Val) substitutions over the N-terminal His41 (Arg41-->His) substitution, and the intraallelic dominance of Thr45 (Arg45-->Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88-->Leu) and Arg256 (Pro256-->Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Val288 (Asp288-->Val), and the double mutant was susceptible to activation by benzoates.(ABSTRACT TRUNCATED AT 250 WORDS)

Transvection in the Drosophila Ultrabithorax gene: a Cbx1 mutant allele induces ectopic expression of a normal allele in trans.

In wild-type Drosophila melanogaster larvae, the Ultrabithorax (Ubx) gene is expressed in the haltere imaginal discs but not in the majority of cells of the wing imaginal discs. Ectopic expression of the Ubx gene in wing discs can be elicited by the presence of Contrabithorax (Cbx) gain-of-function alleles of the Ubx gene or by loss-of-function mutations in Polycomb (Pc) or in other trans-regulatory genes which behave as repressors of Ubx gene activity. Several Ubx loss-of-function alleles cause the absence of detectable Ubx proteins (UBX) or the presence of truncated UBX lacking the homeodomain. We have compared adult wing phenotypes with larval wing disc UBX patterns in genotypes involving double mutant chromosomes carrying in cis one of those Ubx mutations and the Cbx1 mutation. We show that such double mutant genes are (1) active in the same cells in which the single mutant Cbx1 is expressed, although they are unable to yield functional proteins, and (2) able to induce ectopic expression of a normal homologous Ubx allele in a part of the cells in which the single mutant Cbx1 is active. That induction is conditional upon pairing of the homologous chromosomes (the phenomenon known as transvection), and it is not mediated by UBX. Depletion of Pc gene products by Pc3 mutation strongly enhances the induction phenomenon, as shown by (1) the increase of the number of wing disc cells in which induction of the homologous allele is detectable, and (2) the induction of not only a paired normal allele but also an unpaired one.