Levels In Heterozygotes Research Articles

Heterotypic Stressors Unmask Behavioral Influences of PMAT Deficiency in Mice.

Certain life stressors having enduring physiological and behavioral consequences, in part by eliciting dramatic signaling shifts in monoamine neurotransmitters. High monoamine levels can overwhelm selective transporters like the serotonin transporter. This is when polyspecific transporters like plasma membrane monoamine transporter (PMAT, Slc29a4) are hypothesized to contribute most to monoaminergic signaling regulation. Here, we employed two distinct counterbalanced stressors-fear conditioning and swim stress-in mice to systematically determine how reductions in PMAT function affect heterotypic stressor responsivity. We hypothesized that male heterozygotes would exhibit augmented stressor responses relative to female heterozygotes. Decreased PMAT function enhanced context fear expression, an effect unexpectedly obscured by a sham stress condition. Impaired cued fear extinction retention and enhanced context fear expression in males were conversely unmasked by a sham swim condition. Abrogated corticosterone levels in male heterozygotes that underwent swim stress after context fear conditioning did not map onto any measured behaviors. In sum, male heterozygous mouse fear behaviors proved malleable in response to preceding stressor or sham stress exposure. Combined, these data indicate that reduced male PMAT function elicits a form of stress-responsive plasticity. Future studies should assess how PMAT is differentially affected across sexes and identify downstream consequences of the stress-shifted corticosterone dynamics.

Identification of Safe and Effective Intravenous Dose of AAVrh.10hFXN to Treat the Cardiac Manifestations of Friedreich's Ataxia.

Friedreich's ataxia (FA) is a life-threatening autosomal recessive disorder characterized by neurological and cardiac dysfunction. Arrhythmias and heart failure are the main cause of premature death. From prior studies in murine models of FA, adeno-associated virus encoding the normal human frataxin gene (AAVrh.10hFXN) effectively treated the cardiac manifestations of the disease. However, the therapeutic dose window is limited by high level of human frataxin (hFXN) gene expression associated with toxicity. As a therapeutic goal, since FA heterozygotes have no clinical manifestations of FA, we estimated the level of frataxin (FXN) necessary to convert the heart of a homozygote to that of a heterozygote. In noncardiac cells, FA heterozygotes have 30-80% of normal FXN levels (17.7-47.2 ng/mg, average 32.5 ng/mg) and FA homozygotes 2-30% normal levels (1.2-17.7 ng/mg, average 9.4 ng/mg). Therefore, an AAV vector would need to augment endogenous in an FA homozygote by >8.3 ng/mg. To determine the required dose of AAVrh.10hFXN, we administered 1.8 × 1011, 5.7 × 1011, or 1.8 × 1012 gc/kg of AAVrh.10hFXN intravenously (IV) to muscle creatine kinase (mck)-Cre conditional knockout Fxn mice, a cardiac and skeletal FXN knockout model. The minimally effective dose was 5.7 × 1011 gc/kg, resulting in cardiac hFXN levels of 6.1 ± 4.2 ng/mg and a mild (p < 0.01 compared with phosphate-buffered saline controls) improvement in mortality. A dose of 1.8 × 1012 gc/kg resulted in cardiac hFXN levels of 33.7 ± 6.4 ng/mg, a significant improvement in ejection fraction and fractional shortening (p < 0.05, both comparisons) and a 21.5% improvement in mortality (p < 0.001). To determine if the significantly effective dose of 1.8 × 1012 gc/kg could achieve human FA heterozygote levels in a large animal, this dose was administered IV to nonhuman primates. After 12 weeks, the vector-expressed FXN in the heart was 17.8 ± 4.9 ng/mg, comparable to the target human levels. These data identify both minimally and significantly effective therapeutic doses that are clinically relevant for the treatment of the cardiac manifestations of FA.

Molecular characterisation of Hb Akron [β52 (D3) Asp→Val] combined with thalassaemia in a Chinese family

AimsHb Akron (HBB:c.158A>T) is a rare β-chain variant and many characteristics about its clinical features still remain unclear. In this study, we aimed to explore the molecular and haematological characterisations...

Association Between MTHFR Gene Common Variants, Serum Homocysteine, and Risk of Early-Onset Coronary Artery Disease: A Case-Control Study.

The common variants of the methylenetetrahydrofolate reductase (MTHFR) gene are related to the activity of the MTHFR enzyme and the concentrations of blood homocysteine (Hcy). This study was designed to investigate the associations of MTHFR in Chinese populations with early-onset coronary artery disease (EOCAD). The two common variants of the MTHFR gene were genotyped in 875 EOCAD patients and 956 controls using PCR, followed by direct sequencing of the PCR product. Serum levels of Hcy were measured using an automatic biochemistry analyzer. A significant association between the MTHFR-677C/T variant and the risk of EOCAD was detected in CC versus TT (odds ratio (OR) 1.456, 95% confidence interval (CI) 1.120-1.892), dominant genetic model (OR 1.266, 95% CI 1.027-1.546), and recessive genetic model (OR 1.306, 95% CI 1.040-1.639). Hcy was most abundant in TT genotype (18.31 ± 7.22μmol/L), least abundant in CC genotype (11.37 ± 5.23μmol/L), and detectable at intermediate levels in heterozygotes (15.25 ± 6.58μmol/L). Elevated serum Hcy levels were an independent risk factor for EOCAD (ORadjust 1.431, 95% CI 1.135-1.763). Our findings indicated that the T allele of -677C/T MTHFR variant predisposes to high levels of Hcy, and that the T allele is an important risk factor for EOCAD in the Chinese population.

The role of nitrite in muscle function, susceptibility to contraction injury, and fatigability in sickle cell mice

Sickle cell disease (SCD) patients can have limited exercise capacity and muscle dysfunction characterized by decreased force, atrophy, microvascular abnormalities, fiber distribution changes, and skeletal muscle energetics abnormalities. Growing evidence suggests that in SCD there is alteration in nitric oxide (NO) availability/signaling and that nitrate/nitrite can serve as a NO reservoir and enhance muscle performance. Here, we examined effects of nitrite on muscle strength, exercise capacity, and on contractile properties of fast-(extensor digitorum longus, EDL) and slow-twitch (soleus) muscles in SCD mice. Compared to controls, homozygotes (sickling) had decreased grip strength, impaired wheel running performance, and decreased muscle mass of fast-twitch, but not slow-twitch muscle. Nitrite treatment yielded increases in nitrite plasma levels in controls, heterozygotes, and homozygotes but decreases in muscle nitrite levels in heterozygotes and homozygotes. Regardless of genotype, nitrite yielded increases in grip strength, which were coupled with increases in specific force in EDL, but not in soleus muscle. Further, nitrite increased EDL, but not soleus, fatigability in all genotypes. Conversely, in controls, nitrite decreased, whereas in homozygotes, it increased EDL susceptibility to contraction-induced injury. Interestingly, nitrite yielded no changes in distances ran on the running wheel. These differential effects of nitrite in fast- and slow-twitch muscles suggest that its ergogenic effects would be observed in high-intensity/short exercises as found with grip force increases but no changes on wheel running distances. Further, the differential effects of nitrite in homozygotes and control animals suggests that sickling mice, which have altered NO availability/signaling, handle nitrite differently than do control animals.

Analysis and dissociation of anti-HIV effects of shRNA to CCR5 and the fusion inhibitor C46.

The gene therapeutic Cal-1 comprises the anti-HIV agents: (i) sh5, a short hairpin RNA to CCR5 that down-regulates CCR5 expression and (ii) maC46 (C46), a peptide that inhibits viral fusion with the cell membrane. These constructs were assessed for inhibition of viral replication and selective cell expansion in a number of settings. HIV replication, selective outgrowth and cell surface viral binding were analysed with a single cycle infection assay of six pseudotyped HIV strains and a static and longitudinal passaging of MOLT4/CCR5 cells with HIV. Pronase digestion of surface virus and fluorescence microscopy assessed interactions between HIV virions and transduced cells. Cal-1 reduced CCR5 expression in peripheral blood mononuclear cells to CCR5Δ32 heterozygote levels. Even low level transduction resulted in significant preferential expansion in MOLT4/CCR5 gene-containing cells over a 3-week HIV challenge regardless of viral suppression [12.5% to 47.0% (C46), 46.7% (sh5), 62.2% (Dual), respectively]. The sh5 and Dual constructs at > 95% transduction also significantly suppressed virus to day 12 in the passage assay and all constructs, at varying percentage transduction inhibited virus in static culture. No escape mutations were present through 9weeks of challenge. The Dual construct significantly suppressed infection by a panel of CCR5-using viruses, with its efficacy being independently determined from the single constructs. Dual and sh5 inhibited virion internalisation, as determined via pronase digestion of surface bound virus, by 70% compared to 13% for C46. The use of two anti-HIV genes allows optimal preferential survival and inhibition of HIV replication, with the impact on viral load being dependent on the percentage of gene marked cells.

Novel Association of a F11 Variant with Venous Thromboembolism in a Chinese Han Population

Background: Coagulation factor XI (FXI) is essential for normal function of the intrinsic pathway of blood coagulation. Nevertheless, high coagulation factor XI (FXI) levels were related to an increase in the risk of venous thromboembosis. The aim of the present study was to investigate whether any genetic variant in the factor XI gene (F11) was associated with susceptibility to venous thromboembosis in the Chinese Han population.Methods: All exons and the 5'- and 3'-untranslated regions of F11 were initially sequenced to identify informative variants. Potential abnormal variants were analyzed in a population of 1531 venous thrombosis patients and 1343 healthy controls. Finally, functional studies were performed to evaluate the effects of the variant.Results: A mutation c.539A>G(rs190283743) was identified and harbored a high predisposition to venous thrombosis (adjusted OR = 1.861, 95%CI = 1.221-2.836). The plasma factor XI activity and antigen levels in heterozygotes were increased.Conclusions: Our results suggested that the novel F11variant is a possible genetic determinant of an increased risk venous thrombosis of and increased coagulant activity of factor XI. DisclosuresNo relevant conflicts of interest to declare.

Methylation diet and methyl group genetics in risk for adenomatous polyp occurrence

PurposeThe aim of this study is to explore whether a methylation diet influences risk for adenomatous polyps (AP) either independently, or interactively with one-carbon metabolism-dependent gene variants, and whether such a diet modifies blood homocysteine, a biochemical phenotype closely related to the phenomenon of methylation. Methods249 subjects were examined using selective fluorescence, PCR and food frequency questionnaire to determine homocysteine, nine methylation-related gene polymorphisms, dietary methionine, 5-methyltetrahydrofolate, vitamins B6 and B12. Results1). Both dietary methionine and 5-methyltetrahydrofolate intake are significantly associated with plasma homocysteine. 2). Dietary methionine is related to AP risk in 2R3R-TS wildtype subjects, while dietary B12 is similarly related to this phenotype in individuals heterozygous for C1420T-SHMT, A2756G-MS and 844ins68-CBS, and in those recessive for 2R3R-TS. 3). Dietary methionine has a marginal influence on plasma homocysteine level in C1420T-SHMT heterozygotes, while B6 exhibits the same effect on homocysteine in C776G-TCN2 homozygote recessive subjects. Natural 5-methyltetrahydrofolate intake is interesting: Wildtype A1298C-MTHFR, heterozygote C677T-MTHFR, wildtype A2756G-MS and recessive A66G-MSR individuals all show a significant reciprocal association with homocysteine. 4). Stepwise regression of all genotypes to predict risk for AP indicated A2756G-MS and A66G-MSR to be most relevant (p=0.0176 and 0.0408 respectively). Results were corrected for age and gender. ConclusionA methylation diet influences methyl group synthesis in the regulation of blood homocysteine level, and is modulated by genetic interactions. Methylation-related nutrients also interact with key genes to modify risk of AP, a precursor of colorectal cancer. Independent of diet, two methylation-related genes (A2756G-MS and A66G-MSR) were directly associated with AP occurrence.

Interactions between the chromatin remodeller CHD1 and the spliceosome are critical for hematopoietic stem and progenitor cell emergence

Defects in hematopoietic stem cells (HSCs) lead to hematological diseases, such as myelodysplastic syndromes (MDS) and leukemia. Recurrent mutations in spliceosomal components were recently identified in these diseases, suggesting an underappreciated role for splicing regulation in HSC biology. Based on this hypothesis, we explored the importance of splicing on hematopoietic stem and progenitor cell (HSPC) specification during zebrafish development. Zebrafish mutants in U2 snRNP components, specifically sf3a3hi1950 and sf3b1hi3394, have diminished runx1 expression within their aorta by 28 hours post fertilization (hpf), while expression of the endothelial marker flk1/kdrl is mostly unaffected, indicating a defect at the level of HSPC formation. Using these mutants, we defined chd1 as a genetic interaction partner with the spliceosome that is critical for HSPC emergence. CHD1 (Chromodomain Helicase DNA Binding Protein 1) is a chromatin remodeler that interacts with SF3A and SF3B spliceosomal complexes in human cells and is needed for their recruitment to actively transcribed genes. Transient morpholino-mediated knockdown of chd1 in zebrafish embryos has no effect on HSPC development. Similarly, embryos heterozygous for sf3a3hi1950 have no noticeable hematopoietic phenotype, however, in combination, diminished chd1 levels in sf3a3hi1950 heterozygotes leads to a significant decline in HSPC levels. These results demonstrate a clear in vivo connection between splicing and epigenetic regulation within HSPCs. As these two processes are the most frequently mutated in MDS, our data might also have implications for understanding the origins of MDS at the HSC level. Defects in hematopoietic stem cells (HSCs) lead to hematological diseases, such as myelodysplastic syndromes (MDS) and leukemia. Recurrent mutations in spliceosomal components were recently identified in these diseases, suggesting an underappreciated role for splicing regulation in HSC biology. Based on this hypothesis, we explored the importance of splicing on hematopoietic stem and progenitor cell (HSPC) specification during zebrafish development. Zebrafish mutants in U2 snRNP components, specifically sf3a3hi1950 and sf3b1hi3394, have diminished runx1 expression within their aorta by 28 hours post fertilization (hpf), while expression of the endothelial marker flk1/kdrl is mostly unaffected, indicating a defect at the level of HSPC formation. Using these mutants, we defined chd1 as a genetic interaction partner with the spliceosome that is critical for HSPC emergence. CHD1 (Chromodomain Helicase DNA Binding Protein 1) is a chromatin remodeler that interacts with SF3A and SF3B spliceosomal complexes in human cells and is needed for their recruitment to actively transcribed genes. Transient morpholino-mediated knockdown of chd1 in zebrafish embryos has no effect on HSPC development. Similarly, embryos heterozygous for sf3a3hi1950 have no noticeable hematopoietic phenotype, however, in combination, diminished chd1 levels in sf3a3hi1950 heterozygotes leads to a significant decline in HSPC levels. These results demonstrate a clear in vivo connection between splicing and epigenetic regulation within HSPCs. As these two processes are the most frequently mutated in MDS, our data might also have implications for understanding the origins of MDS at the HSC level.

A short antisense oligonucleotide ameliorates symptoms of severe mouse models of spinal muscular atrophy.

Recent reports underscore the unparalleled potential of antisense-oligonucleotide (ASO)-based approaches to ameliorate various pathological conditions. However, in vivo studies validating the effectiveness of a short ASO (<10-mer) in the context of a human disease have not been performed. One disease with proven amenability to ASO-based therapy is spinal muscular atrophy (SMA). SMA is a neuromuscular disease caused by loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Correction of aberrant splicing of the remaining paralog, SMN2, can rescue mouse models of SMA. Here, we report the therapeutic efficacy of an 8-mer ASO (3UP8i) in two severe models of SMA. While 3UP8i modestly improved survival and function in the more severe Taiwanese SMA model, it dramatically increased survival, improved neuromuscular junction pathology, and tempered cardiac deficits in a new, less severe model of SMA. Our results expand the repertoire of ASO-based compounds for SMA therapy, and for the first time, demonstrate the in vivo efficacy of a short ASO in the context of a human disease.

Changes in levels of peripheral hormones controlling appetite are inconsistent with hyperphagia in leptin-deficient subjects

Congenital leptin deficiency, a rare genetic disorder due to a homozygous mutation in the leptin gene (LEP), is accompanied by extreme obesity and hyperphagia. A number of gastrointestinal hormones have been shown to critically regulate food intake but their physiological role in hyperphagic response in congenital leptin deficiency has not been elucidated. This study is the first to evaluate the fasting and postprandial profiles of gut-derived hormones in homozygous and heterozygous carriers of LEP mutation. The study subjects from two consanguineous families consisted of five homozygous and eight heterozygous carriers of LEP mutation, c.398delG. Ten wild-type normal-weight subjects served as controls. Fasting and 1-h postprandial plasma ghrelin, glucagon-like peptide (GLP) 1, peptide YY (PYY), leptin and insulin levels were measured by immunoassays. Fasting plasma ghrelin levels in homozygotes remained remarkably unchanged following food consumption (P = 0.33) in contrast to a significant decline in heterozygous (P < 0.03) and normal (P < 0.02) subjects. A significant postprandial increase in PYY was observed in heterozygous (P < 0.02) and control subjects (P < 0.01), but not in the homozygous group (P = 0.22). A postprandial rise in GLP-1 levels was significant (P < 0.02) in all groups. Interestingly, fasting leptin levels in heterozygotes were not significantly different from controls and did not change significantly following meal. Our results demonstrate that gut hormones play little or no physiological role in driving the hyperphagic response of leptin-deficient subjects. In contrast, fasting and postprandial levels of gut hormones in heterozygous mutation carriers were comparable to those of normal-weight controls.

Transgenic mouse models of the truncated platelet integrin β3 cytoplasmic tail established by stem cell transplantation

This study was purpose to establish the transgenic mouse models of the truncated platelet integrin β3 by retrovirus-infected hematopoietic stem cells (HSCs) transplantation and to provide the basis for further study of the role of integrin β3 cytoplasmic domain in platelet bi-directional signaling pathways. Wild-type β3, β3-Δ759 (R(760) GT(762) truncated β3) and β3-Δ754 (T(755) NITYRGT(762) truncated β3) cDNAs were subcloned into MSCV MigR1 retroviral vector bearing a GFP gene and packaged into infective retrovirus with BOSC23 cell strain. The bone marrow HSCs of the β3 deficient mice were infected by the retroviruses, and transplanted into lethally-irradiated wild type C57BL/6 mice. GFP positive rate and surface β3 expression of the recipients' platelets at 6 to 8 weeks after transplantation were detected by flow cytometry to evaluate the transgenic efficiency. The results showed that four kinds of transgenic mouse models including vector, wild-type β3, β3-Δ759 and β3-Δ754 were established successfully. GFP positive rates of transgenic mouse platelets ranged from 18% to 66% and the β3 expression of transgenic mouse reached heterozygote (β3(+/-) level of mouse). It is concluded that establishment of transgenic mouse models mediated by retrovirus-infected HSCs transplantation is a feasible, fast, and high throughput transgenic approach and laid a solid foundation for further research on the role of integrin β3 cytoplasmic domain for bi-directional signaling of platelets in vivo, and for the gene therapy of platelet disorders.

Aquaporin 11 insufficiency modulates kidney susceptibility to oxidative stress

Aquaporin 11 (AQP11) is a newly described member of the protein family of transport channels. AQP11 associates with the endoplasmic reticulum (ER) and is highly expressed in proximal tubular epithelial cells in the kidney. Previously, we identified and characterized a recessive mutation of the highly conserved Cys227 to Ser227 in mouse AQP11 that caused proximal tubule (PT) injury and kidney failure in mutant mice. The current study revealed induction of ER stress, unfolded protein response, and apoptosis as molecular mechanisms of this PT injury. Cys227Ser mutation interfered with maintenance of AQP11 oligomeric structure. AQP11 is abundantly expressed in the S1 PT segment, a site of major renal glucose flux, and Aqp11 mutant mice developed PT-specific mitochondrial injury. Glucose increased AQP11 protein expression in wild-type kidney and upregulation of AQP11 expression by glucose in vitro was prevented by phlorizin, an inhibitor of sodium-dependent glucose transport across PT. Total AQP11 levels in heterozygotes were higher than in wild-type mice but were not further increased in response to glucose. In Aqp11 insufficient PT cells, glucose potentiated increases in reactive oxygen species (ROS) production. ROS production was also elevated in Aqp11 mutation carriers. Phenotypically normal mice heterozygous for the Aqp11 mutation repeatedly treated with glucose showed increased blood urea nitrogen levels that were prevented by the antioxidant sulforaphane or by phlorizin. Our results indicate an important role for AQP11 to prevent glucose-induced oxidative stress in proximal tubules.

A Novel DNM2 Mutation Displaying Embryonic Lethality and Impaired Transferrin Uptake Identified in a Mouse ENU Mutagenesis Screen for Genes Perturbing Erythropoiesis

AbstractAbstract 608Forward-genetic screens have become a powerful method to study the pathogenesis of human disease and gene function. Chemical mutagenesis in mice using the mutagen, N-ethyl N-nitrosourea (ENU), has shown to be highly successful in elucidating novel genes or alleles in a variety of biological pathways, describing new functions of existing genes, and establishing mouse models that accurately recapitulate human disease. Advances in mapping strategies and deep sequencing technologies has dramatically simplified mutation detection, making ENU mutagenesis screens a feasible tool to study specific organ systems. To identify novel alleles regulating erythropoiesis, our laboratory has undertaken a dominant ENU mutagenesis screen. In this screen, the G1 progeny were screened at seven weeks of age for abnormalities in red cell indices (MCV, MCH, and HCT) using an automated hematological analyser. Here, we describe the identification of mice with a missense mutation of the large GTPase Dynamin 2 (DNM2) leading to an amino acid substitution V235G, predicted to lie within the nucleotide binding pocket for GTP. Western blot analysis for DNM2 protein revealed 50% protein levels in heterozygotes, suggesting that the point mutation leads to loss of protein rather than a dominant negative effect. Inherited DNM2 mutations are associated with autosomal dominant Charcot Tooth Myopathy (CTM) and Centronuclear Myopathy (CNM), but no recognised blood disorders. Heterozygous DNM2V235G displayed hypochromic, microcytic anemia – HGB (15 g/dl compared to 16.5 g/dl in wild type mice), MCV (41.3 fl compared to 45.6 fl in wild type mice) and MCH (12.7 pg compared to 14.5 pg in wild type mice), but no obvious neuropathy or myopathy. Homozygosity was lethal before embryonic (E) day 8.5. DNM2 is an essential component in clathrin-mediated endocytosis, which is required for uptake of transferrin into red cells for incorporation of heme. Accordingly, endocytosis assays for transferrin uptake by FACS and confocal microscopy revealed reduced uptake in heterozygotes, explaining the microcytic hypochromic anemia. Western blot analysis for ferritin demonstrated reduced cellular ferritin, indicating cellular iron deficiency. Thus, this mouse model provides the first in vivo evidence that haplo-insufficiency of DNM2 can lead to iron deficiency anemia. Disclosures:No relevant conflicts of interest to declare.

Genetic Background Analysis of Protein C Deficiency Demonstrates a Recurrent Mutation Associated with Venous Thrombosis in Chinese Population

BackgroundProtein C (PC) is one of the most important physiological inhibitors of coagulation proteases. Hereditary PC deficiency causes a predisposition to venous thrombosis (VT). The genetic characteristics of PC deficiency in the Chinese population remain unknown.MethodsThirty-four unrelated probands diagnosed with hereditary PC deficiency were investigated. PC activity and antigen levels were measured. Mutation analysis was performed by sequencing the PROC gene. In silico analyses, including PolyPhen-2, SIFT, multiple sequence alignment, splicing prediction, and protein molecular modeling were performed to predict the consequences of each variant identified. One recurrent mutation and its relative risk for thrombosis in relatives were analyzed in 11 families. The recurrent mutation was subsequently detected in a case (VT patients)-control study, and the adjusted odds ratio (OR) for VT risk was calculated by logistic regression analysis.ResultsA total of 18 different mutations, including 12 novel variants, were identified. One common mutation, PROC c.565C>T (rs146922325:C>T), was found in 17 of the 34 probands. The family study showed that first-degree relatives bearing this variant had an 8.8-fold (95%CI = 1.1–71.6) increased risk of venous thrombosis. The case-control (1003 vs. 1031) study identified this mutation in 5.88% patients and in 0.87% controls, respectively. The mutant allele conferred a high predisposition to venous thrombosis (adjusted OR = 7.34, 95%CI = 3.61–14.94). The plasma PC activity and antigen levels in heterozygotes were 51.73±6.92 U/dl and 75.17±4.84 U/dl, respectively.ConclusionsThis is the first study on the genetic background of PC deficiency in the Chinese population. The PROC c.565C>T mutation is the most frequent cause of PC deficiency as well as a prevalent risk factor for VT in Chinese individuals. The inclusion of this variant in routine thrombophilic detection may improve the diagnosis and prevention of venous thrombosis.

β-Cells with relative low HIMP1 overexpression levels in a transgenic mouse line enhance basal insulin production and hypoxia/hypoglycemia tolerance.

Rodent pancreatic β-cells that naturally lack hypoglycemia/hypoxia inducible mitochondrial protein 1 (HIMP1) are susceptible to hypoglycemia and hypoxia influences. A linkage between the hypoglycemia/hypoxia susceptibility and the lack of HIMP1 is suggested in a recent study using transformed β-cells lines. To further illuminate this linkage, we applied mouse insulin 1 gene promoter (MIP) to control HIMP1-a isoform cDNA and have generated three lines (L1 to L3) of heterozygous HIMP1 transgenic (Tg) mice by breeding of three founders with C57BL/6J mice. In HIMP1-Tg mice/islets, we performed quantitative polymerase chain reaction (PCR), immunoblot, histology, and physiology studies to investigate HIMP1 overexpression and its link to β-cell function/survival and body glucose homeostasis. We found that the HIMP1 level increased steadily in β-cells of L1 to L3 heterozygous HIMP1-Tg mice. HIMP1 overexpression at relatively lower levels in L1 heterozygotes results in a negligible decline in blood glucose concentrations and an insignificant elevation in blood insulin levels, while HIMP1 overexpression at higher levels are toxic, causing hyperglycemia in L2/3 heterozygotes. Follow-up studies in 5–30-week-old L1 heterozygous mice/islets found that HIMP1 overexpression at relatively lower levels in β-cells has enhanced basal insulin biosynthesis, basal insulin secretion, and tolerances to low oxygen/glucose influences. The findings enforced the linkage between the hypoglycemia/hypoxia susceptibility and the lack of HIMP1 in β-cells, and show a potential value of HIMP1 overexpression at relatively lower levels in modulating β-cell function and survival.

Igf2 ligand dependency of Pten+/− developmental and tumour phenotypes in the mouse

The tumour suppressor PTEN is a key negative regulator of the PI3K-Akt pathway, and is frequently either reduced or lost in human tumours. Murine genetic studies have confirmed that reduction of Pten promotes tumourigenesis in multiple organs, and demonstrated dependency of tumour development on the activation of downstream components such as Akt. Insulin-like growth factors (IGFs) act via IGF1R to activate the PI3K-Akt pathway, and are commonly upregulated in cancer. A context-dependent interplay between IGFs and PTEN exists in normal tissue and tumours; increased IGF2 ligand supply induces Pten expression creating an autoregulatory negative feedback loop, whereas complete loss of PTEN may either cooperate with IGF overexpression in tumour promotion, or result in desensitisation to IGF ligand. However, it remains unknown whether neoplasia associated with Pten loss is dependent on upstream IGF ligand supply in vivo. We evaluated this by generation of Pten+/− mice with differing allelic dosage of Igf2, an imprinted gene encoding the potent embryonic and tumour growth factor Igf2. We show that biallelic Igf2 supply potentiates a previously unreported Pten+/− placental phenotype and results in strain-dependent cardiac hyperplasia and neonatal lethality. Importantly, we also show that the effects of Pten loss in vivo are modified by Igf2 supply, as lack of Igf2 results in extended survival and delayed tumour development while biallelic supply is associated with reduced lifespan and accelerated neoplasia in females. Furthermore, we demonstrate that reduction of PTEN protein to heterozygote levels in human MCF7 cells is associated with increased proliferation in response to IGF2, and does not result in desensitisation to IGF2 signalling. These data indicate that the effects of Pten loss at heterozygote levels commonly observed in human tumours are modified by Igf2 ligand, and emphasise the importance of the evaluation of upstream pathways in tumours with Pten loss.

CBG Santiago: A Novel CBG Mutation

Corticosteroid-binding globulin (CBG; SERPIN A6) gene mutations are rare; only four mutations have been described, often in association with fatigue and chronic pain, albeit with incomplete penetrance. We report a kindred with a novel SERPINA6 mutation. The proband, a 9-yr-old male, had excessive postexertional fatigue, weakness, and migraine. Investigations revealed low morning and ACTH-stimulated peak cortisol levels. SERPIN A6 sequencing detected a novel exon 2 single base deletion (c.13delC) leading to a frameshift generating a stop codon within the signal peptide coding region (p.Leu5CysfsX26) and 50% reduced CBG levels in heterozygotes. The patient's father and two sisters share the mutation. Symptom expression within the family may have been modified by a polymorphic CBG allele (c.735G>T). Exogenous hydrocortisone had no effect on the fatigue. This report documents the fifth CBG gene mutation in humans and the second causing major effects on CBG levels. Individuals with low CBG levels may be misdiagnosed as having secondary hypocortisolism. The association with fatigue and idiopathic pain is again noted and may relate to altered stress system function. Variability of the phenotype may relate to other genetic variations of the CBG gene or environmental factors.

P02-227 - Imprinting in the schizophrenia candidate gene GABRB2

Imprinting, characterized by unequal expression of the offspring's genes in a parent-of-origin dependent manner, has been functionally implicated in brain development and in psychiatric disorders. In this study, unambiguous distortion in paternal but not maternal transmission of the disease-associated single-nucleotide polymorphism (SNP) rs6556547 (T/G) clearly indicated the presence of parent-of-origin effect (POE) in the GABAA receptor β2 subunit gene (GABRB2). ‘Flipping’ of allelic mRNA expression in heterozygotes of SNP rs2229944 (C/T) and the observed two-tiered distribution of mRNA expression levels in heterozygotes of the disease-associated SNP rs1816071 (G/A) furnished important support for the occurrence of imprinting at GABRB2. Imprinting in effect introduced heterozygotes from different parents-of-origin endowed with dissimilar mRNA expression capabilities. The deficit of upper-tiered expressions accounted for the lowered mRNA expression levels in the schizophrenic heterozygotes. This pointed to the necessity of differentiating between two kinds of heterozygotes of different parental origins in disease association studies on GABRB2. Bisulfite sequencing revealed hypermethylation in the neighborhood of SNP rs1816071, and methylation differences between controls and schizophrenia patients. Notably, allele-specific methylation was observed at the disease-associated SNPs rs6556547 and rs1816071. These findings raised the possibility that CpG methylation status of these sites could have an impact on the expression of GABRB2 and the risk of schizophrenia. Furthermore, the occurrence of imprinting and allele-specific methylation in the schizophrenia candidate gene GABRB2 was compatible with the epigenetic hypothesis for schizophrenia pathophysiology, thereby calling for the need to explore the role of epigenetic factors in mediating susceptibility to schizophrenia.

Glutathione S-transferase copy number variation alters lung gene expression

The glutathione S-transferase (GST) enzymes catalyse the conjugation of xenobiotics to glutathione. Based on reports that inherited copy number variations (CNVs) modulate some GST gene expression levels, and that the small airway epithelium (SAE) and alveolar macrophages (AMs) are involved early in the pathogenesis of smoking-induced lung disease, we asked: do germline CNVs modulate GST expression levels in SAE and AMs? Microarrays were used to survey GST gene expression and determine CNVs genotypes in SAE and AMs obtained by bronchoscopy from current smokers and nonsmokers. 26% of subjects were null for both GSTM1 alleles, with reduced GSTM1 mRNA levels seen in both SAE and AMs. 30% of subjects had homozygous deletions of GSTT1, with reduced mRNA levels in both tissues. Interestingly, GSTT2B exhibited homozygous deletion in the blood of 27% of subjects and was not expressed in SAE in the remainder of subjects, but was expressed in AMs of heterozygotes and wild-type subjects, proportionate to genotype. These data show a germline CNV-mediated linear relationship of genotype with expression level, suggesting minimal compensation of gene expression levels in heterozygotes, consistent with GST polymorphisms playing a role in the risk of smoking-associated, xenobiotic-induced lung disease.