Over the past decade, the scientific community has begun to recognize the importance of biological sex differences in disease pathology, diagnosis, prevention, and treatment; however, the practice of sex-specific analysis and reporting is not integrated as standard practice by either our federal health agencies or by major medical journals. Despite the reforms of 20 years ago and the general inclusion of women in drug clinical trials, we have yet to see data routinely analyzed and reported by sex. Major journals are not requiring it, and large, publicly available datasets, such as ClinicalTrials.gov, are not systematically collecting and pointing to it. However, federal health databases and medical journals have the potential to impact progress in sex-specific analysis and reporting. We conducted a search on ClinicalTrials.gov for phase III device clinical trials and assessed their practice of sex differences evaluation. Reporting of clinical trial results by sex will maximize scientific value of research investments, significantly impact clinical decision-making, and transform medical care.
DNA double strand breaks are a particularly toxic form of DNA damage and the mammalian cell has evolved an intricate set of responses to repair this type of DNA lesion. A key early event in the DNA damage response (DDR) is ATM phosphorylation of the histone variant H2AX at serine 139 at the site of the DNA break. Phosphorylated S139 H2AX, or γH2AX, forms a docking site for binding of MDC1, leading to sustained recruitment of other DNA repair factors that mediate the repair of the DNA double strand break. Moreover, recruitment of MDC1 to the break site activates cell cycle checkpoints, protecting the cell from replication of damaged DNA templates. While the molecular events leading to DNA double strand break repair have been well described, the deactivating or homeostatic mechanisms following completion of repair remain largely unexplored. Recent publications by our laboratories and the Medema laboratory shed new light on this issue. Both publications showed that the Wild-type p53-Induced Phosphatase 1 (WIP1) directly dephosphorylates γH2AX. WIP1 migrates to the sites of irradiation-induced foci (IRIF), though at a delayed rate relative to MDC1 and mediates γH2AX dephosphorylation, presumably after DNA repair is complete. This prevents recruitment of other repair factors such as MDC1 and 53BP1 to the DNA damage sites and promotes the dissolution of IRIF. In addition, overexpression of WIP1 has a suppressive effect on DNA double strand break repair. Taken together, these reports further implicate WIP1 as a critical homeostatic regulator of the DDR.
Wip1; PPM1D; γH2AX; MDC1; ATM; ATR; DNA double strand break repair
Nesprin-1-giant and nesprin-2-giant regulate nuclear positioning by the interaction of their C-terminal KASH domains with nuclear membrane SUN proteins and their N-terminal calponin-homology domains with cytoskeletal actin. A number of short isoforms lacking the actin-binding domains are produced by internal promotion. We have evaluated the significance of these shorter isoforms using quantitative RT-PCR and western blotting with site-specific monoclonal antibodies. Within a complete map of nesprin isoforms, we describe two novel nesprin-2 epsilon isoforms for the first time. Epsilon isoforms are similar in size and structure to nesprin-1-alpha. Expression of nesprin isoforms was highly tissue-dependent. Nesprin-2-epsilon-1 was found in early embryonic cells, while nesprin-2-epsilon-2 was present in heart and other adult tissues, but not skeletal muscle. Some cell lines lack shorter isoforms and express only one of the two nesprin genes, suggesting that either of the giant nesprins is sufficient for basic cell functions. For the first time, localisation of endogenous nesprin away from the nuclear membrane was shown in cells where removal of the KASH domain by alternative splicing occurs. By distinguishing between degradation products and true isoforms on western blots, it was found that previously-described beta and gamma isoforms are expressed either at only low levels or with a limited tissue distribution. Two of the shortest alpha isoforms, nesprin-1-alpha-2 and nesprin-2-alpha-1, were found almost exclusively in cardiac and skeletal muscle and a highly conserved and alternatively-spliced exon, available in both nesprin genes, was always included in these tissues. These “muscle-specific” isoforms are thought to form a complex with emerin and lamin A/C at the inner nuclear membrane and mutations in all three proteins cause Emery-Dreifuss muscular dystrophy and/or inherited dilated cardiomyopathy, disorders in which only skeletal muscle and/or heart are affected.
Mutation of K-Ras is a frequent oncogenic event in human cancers, particularly cancers of lungs, pancreas, and colon. It remains unclear why some tissues are more susceptible to Ras-induced transformation than others. Here, we globally activated a mutant oncogenic K-Ras allele (K-RasG12D) in mice and examined the tissue-specific effects of this activation on cancer pathobiology, Ras signaling, tumor suppressor, DNA damage, and inflammatory responses. Within 5–6 weeks of oncogenic Ras activation, mice develop oral and gastric papillomas, lung adenomas and hematopoietic hyperproliferation and turn moribund. The oral, gastric and lung pre-malignant lesions display activated Erk1/2 and NF-κB signaling as well as activated tumor suppressor and DNA damage responses. Other organs such as pancreas, liver and small intestine do not exhibit neoplastic progression within six weeks following K-rasG12D activation and do not show a potent tumor suppressor response. Even though robust Erk1/2 signaling is activated in all the tissues examined, the pErk1/2 distribution remains largely cytoplasmic in K-RasG12D refractory tissues (pancreas, liver and intestines) as opposed to a predominantly nuclear localization in K-RasG12D induced neoplasms of lung, oral, and gastric mucosa. The downstream targets of Ras signaling, pElk-1 and c-Myc, are elevated in K-RasG12D induced neoplastic lesions but not in K-RasG12D refractory tissues. We propose that oncogenic K-Ras refractory tissues delay oncogenic progression by spatially limiting the efficacy of Ras/Raf/Erk1/2 signaling, whereas K-Ras responsive tissues exhibit activated Ras/Raf/Erk1/2 signaling, rapidly form pre-malignant tumors, and activate potent anti-tumor responses that effectively prevent further malignant progression.
K-Ras; ERK1/2; Elk-1; nuclear translocation; p53
Dengue virus transmission occurs in both epidemic and endemic cycles across tropical and sub-tropical regions of the world. Incidence is particularly high in much of Southeast Asia, where hyperendemic transmission plagues both urban and rural populations. However, endemicity has not been established in some areas with climates that may not support year-round viral transmission. An understanding of how dengue viruses (DENV) enter these environments and whether the viruses persist in inapparent local transmission cycles is central to understanding how dengue emerges in areas at the margins of endemic transmission. Dengue is highly endemic in tropical southern Vietnam, while increasingly large seasonal epidemics have occurred in northern Viet Nam over the last decade. We have investigated the spread of DENV-1 throughout Vietnam to determine the routes by which the virus enters northern and central regions of the country. Phylogeographic analysis of 1,765 envelope (E) gene sequences from Southeast Asia revealed frequent movement of DENV between neighboring human populations and strong local clustering of viral lineages. Long-distance migration of DENV between human population centers also occurred regularly and on short time-scales, indicating human-mediated viral invasion into northern Vietnam. Human populations in southern Vietnam were found to be the primary source of DENV circulating throughout the country, while central and northern Vietnam acted as sink populations, likely due to reduced connectedness to other populations in the case of the central regions and to the influence of temperature variability on DENV replication and vector survival and competence in the north. Finally, phylogeographic analyses suggested that viral movement follows a gravity model and indicates that population immunity and physical and economic connections between populations may play important roles in shaping patterns of DENV transmission.
Reports from sub-tropical regions of the world suggest a growing risk of introduction and establishment of dengue viruses (DENV) in new locales. Recent dengue epidemics in northern Viet Nam present an opportunity to study how DENV invades and spreads in these environments. The proximity of this region to tropical areas experiencing year-round endemic DENV transmission makes it an ideal site for studying the effects of human population movement and climate on DENV emergence. We performed a phylogenetic analysis using DENV-1 envelope gene sequences from Southeast Asia. We show that DENV are regularly imported into northern and central Viet Nam from southern Vietnam, and that increasingly large seasonal epidemics in the north are caused by newly introduced viruses each year. While tropical Vietnam maintains localized virus populations for multiple years, cool winter temperatures in sub-tropical northern Viet Nam may reduce mosquito populations and virus replication to levels that are not conducive to year-round DENV transmission. Finally, we found that the dispersal of DENV across the region is well-described using human movement and immunity data, and believe that increased epidemiological, entomological, and virological surveillance are needed to understand the processes by which endemic DENV transmission becomes established in new populations.
Oxidative stress is thought to play a significant role in the development and progression of neurodegenerative diseases. Although it is currently considered a hallmark of such processes, the interweaving of a multitude of signaling cascades hinders complete understanding of the direct role of oxidative stress in neurodegeneration. In addition to its extensive use as an aging model, some researchers have turned to the invertebrate model Caenorhabditis elegans (C. elegans) in order to further investigate molecular mediators that either exacerbate or protect against reactive oxygen species (ROS)-mediated neurodegeneration. Due to their fully characterized genome and short life cycle, rapid generation of C. elegans genetic models can be useful to study upstream markers of oxidative stress within interconnected signaling pathways. This report will focus on the roles of C. elegans homologs for the oxidative stress-associated transcription factor Nrf2, as well as the autosomal recessive, early-onset Parkinson’s disease (PD)-associated proteins Parkin, DJ-1, and PINK1, in neurodegenerative processes.
oxidative stress; neurodegeneration; Parkinson’s disease; C. elegans; DJ-1; Parkin; PINK1; Nrf2
Lynch syndrome is an autosomal dominant cancer predisposition syndrome classically caused by germline mutations of the mismatch repair genes, MLH1, MSH2, MSH6 and PMS2. Constitutional epimutations of the MLH1 gene, characterized by soma-wide methylation of a single allele of the promoter and allelic transcriptional silencing, have been identified in a subset of Lynch syndrome cases lacking a sequence mutation in MLH1. We report two individuals with no family history of colorectal cancer who developed that disease at age 18 and 20 years. In both cases, cancer had arisen because of the de novo occurrence of a constitutional MLH1 epimutation and somatic loss-of-heterozygosity of the functional allele in the tumors. We show for the first time that the epimutation in one case arose on the paternally inherited allele. Analysis of 13 tumors from seven individuals with constitutional MLH1 epimutations showed eight tumors had lost the second MLH1 allele, two tumors had a novel pathogenic missense mutation and three had retained heterozygosity. Only 1 of 12 tumors demonstrated the BRAF V600E mutation and 3 of 11 tumors harbored a mutation in KRAS. The finding that epimutations can originate on the paternal allele provides important new insights into the mechanism of origin of epimutations. It is clear that the second hit in MLH1 epimutation-associated tumors typically has a genetic not epigenetic basis. Individuals with mismatch repair–deficient cancers without the BRAF V600E mutation are candidates for germline screening for sequence or methylation changes in MLH1.
colorectal cancer; Lynch syndrome; MLH1 epimutation; microsatellite instability; BRAF
Treatment of congenital adrenal hyperplasia
icroscopic Observation Drug Susceptibility (MODS) has been shown to be an effective and rapid technique for early diagnosis of tuberculosis (TB). Thus far only a limited number of studies evaluating MODS have been performed in children and in extra-pulmonary tuberculosis. This study aims to assess relative accuracy and time to positive culture of MODS for TB diagnosis in children admitted to a general pediatric hospital in Vietnam.
Specimens from children with suspected TB were tested by smear, MODS and Lowenstein-Jensen agar (LJ). 1129 samples from 705 children were analyzed, including sputum (n = 59), gastric aspirate (n = 775), CSF (n = 148), pleural fluid (n = 33), BAL (n = 41), tracheal fluid (n = 45), other (n = 28). 113 TB cases were defined based on the “clinical diagnosis” (confirmed and probable groups) as the reference standard, in which 26% (n = 30) were diagnosed as extra-pulmonary TB. Analysis by patient shows that the overall sensitivity and specificity of smear, LJ and MODS against “clinical diagnosis” was 8.8% and 100%, 38.9% and 100%, 46% and 99.5% respectively with MODS significantly more sensitive than LJ culture (P = 0.02). When analyzed by sample type, the sensitivity of MODS was significantly higher than LJ for gastric aspirates (P = 0.004). The time to detection was also significantly shorter for MODS than LJ (7 days versus 32 days, P<0.001).
ODS is a sensitive and rapid culture technique for detecting TB in children. As MODS culture can be performed at a BSL2 facility and is inexpensive, it can therefore be recommended as a routine test for children with symptoms suggestive of TB in resource-limited settings.
The genetic basis for susceptibility to malaria has been studied widely in African populations but less is known of the contribution of specific genetic variants in Asian populations. We genotyped 67 SNPs in 1030 severe malaria cases and 2840 controls from Vietnam. After data quality control, genotyping data of 956 cases and 2350 controls were analysed for 65 SNPs (3 gender confirmation, 62 positioned in/near 42 malarial candidate genes). 14 SNPs were monomorphic and 2 (rs8078340 and rs33950507) were not in HWE in controls (P<0.01). 7/46 SNPs in 6 genes (ICAM1, IL1A, IL17RC, IL13, LTA and TNF) were associated with severe malaria, with 3/7 SNPs in the TNFA/LTA region . Genotype phenotype correlations between SNPs and clinical parameters revealed that genotypes of rs708567 (IL17RC) correlate with parasitemia (P=0.028, r2=0.0086), with GG homozygotes having the lowest parasite burden. Additionally, rs708567 GG homozygotes had a decreased risk of severe malaria [P=0.007, OR=0.78 (95% CI; 0.65-0.93)] and death [P=0.028, OR=0.58 (95% CI; 0.37-0.93)] than those with AA and AG genotypes. In summary, variants in 6 genes encoding adhesion and pro-inflammatory molecules are associated with severe malaria in the Vietnamese. Further replicative studies in independent populations will be necessary to confirm these findings.
severe malaria; SNP; genetic association; ICAM-1; TNF; IL-17RC
Recent genome-wide association studies (GWAS) of late-onset Alzheimer disease (LOAD) identified 9 novel risk loci. Discovery of functional variants within genes at these loci is required to confirm their role in Alzheimer disease (AD). Single nucleotide polymorphisms that influence gene expression (eSNPs) constitute an important class of functional variants. We therefore investigated the influence of the novel LOAD risk loci on human brain gene expression.
We measured gene expression levels in the cerebellum and temporal cortex of autopsied AD subjects and those with other brain pathologies (∼400 total subjects). To determine whether any of the novel LOAD risk variants are eSNPs, we tested their cis-association with expression of 6 nearby LOAD candidate genes detectable in human brain (ABCA7, BIN1, CLU, MS4A4A, MS4A6A, PICALM) and an additional 13 genes ±100 kb of these SNPs. To identify additional eSNPs that influence brain gene expression levels of the novel candidate LOAD genes, we identified SNPs ±100 kb of their location and tested for cis-associations.
CLU rs11136000 (p = 7.81 × 10−4) and MS4A4A rs2304933/rs2304935 (p = 1.48 × 10−4–1.86 × 10−4) significantly influence temporal cortex expression levels of these genes. The LOAD-protective CLU and risky MS4A4A locus alleles associate with higher brain levels of these genes. There are other cis-variants that significantly influence brain expression of CLU and ABCA7 (p = 4.01 × 10−5–9.09 × 10−9), some of which also associate with AD risk (p = 2.64 × 10−2–6.25 × 10−5).
CLU and MS4A4A eSNPs may at least partly explain the LOAD risk association at these loci. CLU and ABCA7 may harbor additional strong eSNPs. These results have implications in the search for functional variants at the novel LOAD risk loci.
The effects of diverse stresses on promoter selectivity and transcription regulation by the tumor suppressor p53 are poorly understood. We have taken a comprehensive approach to characterizing the human p53 network that includes p53 levels, binding, expression and chromatin changes under diverse stresses. Human osteosarcoma U2OS cells treated with anti-cancer drugs Doxorubicin (DXR) or Nutlin-3 (Nutlin) led to strikingly different p53 gene binding patterns based on chromatin immunoprecipitation with high-throughput sequencing experiments. Although two contiguous RRRCWWGYYY decamers is the consensus binding motif, p53 can bind a single decamer and function in vivo. Although the number of sites bound by p53 was six times greater for Nutlin than DXR, expression changes induced by Nutlin were much less dramatic compared with DXR. Unexpectedly, the solvent dimethylsulphoxide (DMSO) alone induced p53 binding to many sites common to DXR; however, this binding had no effect on target gene expression. Together, these data imply a two-stage mechanism for p53 transactivation where p53 binding only constitutes the first stage. Furthermore, both p53 binding and transactivation were associated with increased active histone modification histone H3 lysine 4 trimethylation. We discovered 149 putative new p53 target genes including several that are relevant to tumor suppression, revealing potential new targets for cancer therapy and expanding our understanding of the p53 regulatory network.
Leucine rich repeat transmembrane protein 3 (LRRTM3) is member of a synaptic protein family. LRRTM3 is a nested gene within α-T catenin (CTNNA3) and resides at the linkage peak for late-onset Alzheimer’s disease (LOAD) risk and plasma amyloid β (Aβ) levels. In-vitro knock-down of LRRTM3 was previously shown to decrease secreted Aβ, although the mechanism of this is unclear. In SH-SY5Y cells overexpressing APP and transiently transfected with LRRTM3 alone or with BACE1, we showed that LRRTM3 co-localizes with both APP and BACE1 in early endosomes, where BACE1 processing of APP occurs. Additionally, LRRTM3 co-localizes with APP in primary neuronal cultures from Tg2576 mice transduced with LRRTM3-expressing adeno-associated virus. Moreover, LRRTM3 co-immunoprecipitates with both endogenous APP and overexpressed BACE1, in HEK293T cells transfected with LRRTM3. SH-SY5Y cells with knock-down of LRRTM3 had lower BACE1 and higher CTNNA3 mRNA levels, but no change in APP. Brain mRNA levels of LRRTM3 showed significant correlations with BACE1, CTNNA3 and APP in ∼400 humans, but not in LRRTM3 knock-out mice. Finally, we assessed 69 single nucleotide polymorphisms (SNPs) within and flanking LRRTM3 in 1,567 LOADs and 2,082 controls and identified 8 SNPs within a linkage disequilibrium block encompassing 5′UTR-Intron 1 of LRRTM3 that formed multilocus genotypes (MLG) with suggestive global association with LOAD risk (p = 0.06), and significant individual MLGs. These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02–0.05). Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of LRRTM3, CTNNA3, APP and BACE1 in humans might influence APP metabolism and ultimately risk of AD.
Previously, we have shown that wild type N-ras (wt N-ras) harbors an anti-malignant effect against mutated Ras and in tumors without Ras mutations. To investigate the molecular bases of this anti-malignant activity, we have studied the potency of this anti-malignant effect in a model system against SV40 large T antigen (SV40T). We show that wild-type N-ras (wt N-ras) counteracts the effects of SV40T in NIH3T3 cells as seen by a decrease in proliferation, anchorage independence and changes in migration. We also show that wt N-ras elicits the same anti-malignant effects in some human tumor cell lines (HT1080 and MDA-MB-231). Through mRNA and microRNA (miRNAs) expression profiling we have identified genes (decorin) and miRNAs (mir-29A, let-7b) modulated by wt N-ras potentially responsible for the anti-malignant effect. Wt N-ras appears to mediate its anti-malignant effect by downregulating some of the targets of the TGFβ pathway and decorin, which are able to reverse the inhibition of migration induced by wt N-ras. Our experiments show that the molecules that mediate the anti-malignant effect by wt N-ras appear to be different from those modulated by transforming N-ras. The components of the pathways modulated by wt N-ras mediating its anti-malignant effects are potential targets for therapeutic intervention in cancer.
Signal transduction pathways; Guanine nucleotide binding proteins and effectors; Ras; anti-malignant effects; migration
By 2009, there were worrying signs from western Cambodia that parasitological responses to artesunate-containing treatment regimens for uncomplicated Plasmodium falciparum malaria were slower than elsewhere which suggested the emergence of artemisinin resistance. Vietnam shares a long land border with Cambodia with a large number of migrants crossing it on a daily basis. Therefore, there is an urgent need to investigate whether there is any evidence of a change in the parasitological response to the artemisinin derivatives in Vietnam.
From August 2010 to May 2011, a randomized controlled clinical trial in uncomplicated falciparum malaria was conducted to compare two doses of artesunate (AS) (2mg/kg/day versus 4 mg/kg/day for three days) followed by dihydroartemisinin-piperaquine (DHA-PPQ) and a control arm of DHA-PPQ. The goal was characterization of the current efficacy of artesunate in southern Vietnam. The primary endpoint of this study was the parasite clearance half-life; secondary endpoints included the parasite reduction ratios at 24 and 48 hours and the parasite clearance time.
166 patients were recruited into the study. The median parasite clearance half-lives were 3.54 (AS 2mg/kg), 2.72 (AS 4mg/kg), and 2.98 hours (DHA-PPQ) (p=0.19). The median parasite-reduction ratio at 24 hours was 48 in the AS 2mg/kg group compared with 212 and 113 in the other two groups, respectively (p=0.02). The proportions of patients with a parasite clearance time of >72 hours for AS 2mg/kg, AS 4mg/kg and DHA-PPQ were 27%, 27%, and 22%, respectively. Early treatment failure occurred in two (4%) and late clinical failure occurred in one (2%) of the 55 patients in the AS 2mg/kg group, as compared with none in the other two study arms. The PCR-corrected adequate clinical and parasitological response (APCR) rates in the three groups were 94%, 100%, and 100% (p=0.04).
This study demonstrated faster P. falciparum parasite clearance in southern Vietnam than in western Cambodia but slower clearance in comparison with historical data from Vietnam. Further studies to determine whether this represents the emergence of artemisinin resistance in this area are needed. Currently, the therapeutic response to DHA-PPQ remains satisfactory in southern Vietnam.
Plasmodium falciparum; Artesunate; Parasite clearance half-life; Parasite reduction ratio; Parasite clearance of >72 hours
Legionella pneumophila is a Gram-negative bacterium that replicates within human alveolar macrophages by evasion of the host endocytic pathway through the formation of a replicative vacuole. Generation of this vacuole is dependent upon the secretion of over 275 effector proteins into the host cell via the Dot/Icm type IVB secretion system (T4SS). The type IV coupling protein (T4CP) subcomplex, consisting of DotL, DotM, DotN, IcmS and IcmW, was recently defined. DotL is proposed to be the T4CP of the L. pneumophila T4SS based on its homology to known T4CPs, which function as inner-membrane receptors for substrates. As a result, DotL is hypothesized to play an integral role(s) in the L. pneumophila T4SS for the engagement and translocation of substrates. To elucidate this role, a genetic approach was taken to screen for dotL mutants that were unable to survive inside host cells. One mutant, dotLY725Stop, did not interact with the type IV adaptor proteins IcmS/IcmW (IcmSW) leading to the identification of an IcmSW-binding domain on DotL. Interestingly, the dotLY725Stop mutant was competent for export of one class of secreted effectors, the IcmSW-independent substrates, but exhibited a specific defect in secretion of IcmSW-dependent substrates. This differential secretion illustrates that DotL requires a direct interaction with the type IV adaptor proteins for the secretion of a major class of substrates. Thus, by identifying a new target for IcmSW, we have discovered that the type IV adaptors perform an additional role in the export of substrates by the L. pneumophila Dot/Icm T4SS.
Many pathogens are able to survive and grow within eukaryotic host cells. One such pathogen, Legionella pneumophila, is able to replicate within macrophages, resulting in a form of pneumonia called Legionnaires' Disease. One key to L. pneumophila's capacity to cause disease is its ability to translocate several hundred proteins into the host cell. These proteins, typically referred to as “effectors”, function to alter the host cell to create a hospitable environment for the bacteria. L. pneumophila effectors are exported by a specialized export apparatus, which is encoded by the dot/icm genes. However, the mechanism of secretion for these substrates is poorly understood. It is known that a subset of these effectors requires assistance from the type IV adaptor proteins IcmS and IcmW for transport out of the bacterium. It has been shown that IcmSW binds adaptor-dependent secreted proteins in the bacterial cytoplasm prior to their export. Here we report that DotL, an inner membrane component of the Dot/Icm secretion system, also binds IcmSW and this interaction is required for the export of adaptor-dependent substrates. This defines a new role for the type IV adaptors IcmSW and furthers our understanding of how Legionella exports substrates into its host cell.
Recently, several genome-wide association studies (GWAS) on bipolar disorder (BPD) suggested novel risk genes. However, only few of them were followed up and further, the specificity of these genes is even more elusive. To address these issues, we genotyped SNPs in ANK3, CACNA1C, CMTM8, DGKH, EGFR, and NPAS3, which were significantly associated with BPD in previous GWAS, in a sample of 380 BPD patients. Replicated SNPs were then followed up in patients suffering from unipolar depression (UPD; n=387) or adult attention-deficit/hyperactivity disorder (aADHD; n=535). While we could not confirm an association of ANK3, CACNA1C, and EGFR with BPD, 10 SNPs in DGKH, CMTM8, and NPAS3 were nominally associated with disease, with two DGKH markers surviving correction for multiple testing. When these were followed up in UPD and aADHD, seven DGKH SNPs were also associated with UPD, while one SNP each in NPAS3 and CMTM8 and four in DGKH were linked to aADHD. Furthermore, a DGKH haplotype consisting of rs994856/rs9525580/rs9525584 GAT was associated with all disorders tested, while the complementary AGC haplotype was protective. The corresponding haploblock spans a 27-kb region covering exons coding for amino acids 65–243, and thus might include functional variants yet to be identified. We demonstrate an association of DGKH with BPD, UPD, and aADHD by applying a two-stage design. These disorders share the feature of mood instability, so that this phenotype might be associated with genetic variation in DGKH.
association; bipolar disorder; depression; adult ADHD; NPAS3; CMTM8; depression; unipolar/bipolar; neurogenetics; signal transduction; biological psychiatry; association; adult ADHD; NPAS3; CMTM8; EGFR
Wildtype p53-Induced Phosphatase 1 (WIP1) is a serine/threonine phosphatase that dephosphorylates proteins in the ataxia telangiectasia mutated (ATM)-initiated DNA damage response pathway. WIP1 may play a homeostatic role in ATM signaling by returning the cell to a normal pre-stress state following completion of DNA repair. To better understand the effects of WIP1 on ATM signaling, we crossed Atm-deficient mice to Wip1-deficient mice and characterized phenotypes of the double knockout progeny. We hypothesized that the absence of Wip1 might rescue Atm deficiency phenotypes. Atm null mice, like ATM-deficient humans with the inherited syndrome ataxia telangiectasia, exhibit radiation sensitivity, fertility defects, and are T-cell lymphoma prone. Most double knockout mice were largely protected from lymphoma development and had a greatly extended lifespan compared to Atm null mice. Double knockout mice had increased p53 and H2AX phosphorylation and p21 expression compared to their Atm null counterparts, indicating enhanced p53 and DNA damage responses. Additionally, double knockout splenocytes displayed reduced chromosomal instability compared to Atm null mice. Finally, doubly null mice were partially rescued from infertility defects observed in Atm null mice. These results indicate that inhibition of WIP1 may represent a useful strategy for cancer treatment in general and A-T patients in particular.
WIP1; PPM1D; ATM; ataxia telangiectasia; p53; thymic lymphoma
Despite numerous preventive strategies on bacterial adhesion, pathogenic biofilm formation remained the major cause of medical device-related infections. Bacterial interference is a promising strategy that uses pre-established biofilms of benign bacteria to serve as live, protective coating against pathogen colonization. However, the application of this strategy to silicone urinary catheters was hampered by low adherence of benign bacteria onto silicone materials. In this work, we present a general method for biofunctionalization of silicone (PDMS) as one of the most widely used materials for biomedical devices. We used mild CO2 plasma to activate PDMS surface followed by simple attachment of generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers to generate an amino-terminated surface that were maintained even after storage in PBS buffer for 36 days. We then covalently attach a carboxy-terminated mannose derivative to the modified PDMS to promote the adherence of benign Escherichia coli 83972 expressing mannose-binding type 1 fimbriae. We demonstrated that dense, stable biofilms of E. coli 83972 could be established within 48 h on the mannose-coated PDMS. Significantly, this benign biofilm reduced the adherence of the uropathogenic Enterococcus faecalis by 104-fold after 72 hours, while the benign bacteria on the unmodified substrate by only 5.5-fold.
Genetic variants that modify brain gene expression may also influence risk for human diseases. We measured expression levels of 24,526 transcripts in brain samples from the cerebellum and temporal cortex of autopsied subjects with Alzheimer's disease (AD, cerebellar n = 197, temporal cortex n = 202) and with other brain pathologies (non–AD, cerebellar n = 177, temporal cortex n = 197). We conducted an expression genome-wide association study (eGWAS) using 213,528 cisSNPs within ±100 kb of the tested transcripts. We identified 2,980 cerebellar cisSNP/transcript level associations (2,596 unique cisSNPs) significant in both ADs and non–ADs (q<0.05, p = 7.70×10−5–1.67×10−82). Of these, 2,089 were also significant in the temporal cortex (p = 1.85×10−5–1.70×10−141). The top cerebellar cisSNPs had 2.4-fold enrichment for human disease-associated variants (p<10−6). We identified novel cisSNP/transcript associations for human disease-associated variants, including progressive supranuclear palsy SLCO1A2/rs11568563, Parkinson's disease (PD) MMRN1/rs6532197, Paget's disease OPTN/rs1561570; and we confirmed others, including PD MAPT/rs242557, systemic lupus erythematosus and ulcerative colitis IRF5/rs4728142, and type 1 diabetes mellitus RPS26/rs1701704. In our eGWAS, there was 2.9–3.3 fold enrichment (p<10−6) of significant cisSNPs with suggestive AD–risk association (p<10−3) in the Alzheimer's Disease Genetics Consortium GWAS. These results demonstrate the significant contributions of genetic factors to human brain gene expression, which are reliably detected across different brain regions and pathologies. The significant enrichment of brain cisSNPs among disease-associated variants advocates gene expression changes as a mechanism for many central nervous system (CNS) and non–CNS diseases. Combined assessment of expression and disease GWAS may provide complementary information in discovery of human disease variants with functional implications. Our findings have implications for the design and interpretation of eGWAS in general and the use of brain expression quantitative trait loci in the study of human disease genetics.
Genetic variants that regulate gene expression levels can also influence human disease risk. Discovery of genomic loci that alter brain gene expression levels (brain expression quantitative trait loci = eQTLs) can be instrumental in the identification of genetic risk underlying both central nervous system (CNS) and non–CNS diseases. To systematically assess the role of brain eQTLs in human disease and to evaluate the influence of brain region and pathology in eQTL mapping, we performed an expression genome-wide association study (eGWAS) in 773 brain samples from the cerebellum and temporal cortex of ∼200 autopsied subjects with Alzheimer's disease (AD) and ∼200 with other brain pathologies (non–AD). We identified ∼3,000 significant associations between cisSNPs near ∼700 genes and their cerebellar transcript levels, which replicate in ADs and non–ADs. More than 2,000 of these associations were reproducible in the temporal cortex. The top cisSNPs are enriched for both CNS and non–CNS disease-associated variants. We identified novel and confirmed previous cisSNP/transcript associations for many disease loci, suggesting gene expression regulation as their mechanism of action. These findings demonstrate the reproducibility of the eQTL approach across different brain regions and pathologies, and advocate the combined use of gene expression and disease GWAS for identification and functional characterization of human disease-associated variants.
Glutathione S-transferase omega-1 and 2 genes (GSTO1, GSTO2), residing within an Alzheimer and Parkinson disease (AD and PD) linkage region, have diverse functions including mitigation of oxidative stress and may underlie the pathophysiology of both diseases. GSTO polymorphisms were previously reported to associate with risk and age-at-onset of these diseases, although inconsistent follow-up study designs make interpretation of results difficult. We assessed two previously reported SNPs, GSTO1 rs4925 and GSTO2 rs156697, in AD (3,493 ADs vs. 4,617 controls) and PD (678 PDs vs. 712 controls) for association with disease risk (case-controls), age-at-diagnosis (cases) and brain gene expression levels (autopsied subjects).
We found that rs156697 minor allele associates with significantly increased risk (odds ratio = 1.14, p = 0.038) in the older ADs with age-at-diagnosis > 80 years. The minor allele of GSTO1 rs4925 associates with decreased risk in familial PD (odds ratio = 0.78, p = 0.034). There was no other association with disease risk or age-at-diagnosis. The minor alleles of both GSTO SNPs associate with lower brain levels of GSTO2 (p = 4.7 × 10-11-1.9 × 10-27), but not GSTO1. Pathway analysis of significant genes in our brain expression GWAS, identified significant enrichment for glutathione metabolism genes (p = 0.003).
These results suggest that GSTO locus variants may lower brain GSTO2 levels and consequently confer AD risk in older age. Other glutathione metabolism genes should be assessed for their effects on AD and other chronic, neurologic diseases.
GSTO genes; Disease risk; Gene expression; Association
Background & Aims
5-FU-based adjuvant chemotherapy does not increase survival times of patients with colorectal tumors with microsatellite instability. We determined the response of patients with colorectal tumors with the CpG island methylator phenotype (CIMP) to 5-FU-based therapy.
We analyzed a population-based cohort of 302 patients with colorectal cancer (CRC) for a median follow-up time of 50.7 months. CIMP status was determined by analysis of the CACNAG1, SOCS1, RUNX3, NEUROG1, and MLH1 promoters; tumors were considered to be CIMP-positive (CIMP+) if at least 3 promoters were methylated.
Tumors from 29.5% (89/302) of patients were CIMP+; this did not influence disease-free survival (log rank=.26). Of tumors of TNM stages II–III (n=196), 32.7% were CIMP+. Among patients with CRC stages II–III who did not receive adjuvant 5-FU chemotherapy, those with CIMP+ tumors had longest times of disease-free survival (log rank=.04); patients with CIMP+ tumors who received chemotherapy had shorter times of disease-free survival (log rank=0.02). In patients with CIMP-negative tumors, adjuvant 5-FU chemotherapy significantly increased time of disease-free survival (log-rank=.00001). However, in patients with CIMP+ tumors, adjuvant 5-FU chemotherapy did not affect time of disease-free survival (log rank=.7). Multivariate analysis showed a significant, independent interaction between 5-FU treatment and CIMP status (hazard ratio [HR]=0.6; 95% confidence interval [CI], .5–.8). Among patients with CIMP+ tumors, adjuvant chemotherapy was not an independent predictor of outcome (HR=0.8; 95% CI, 0.3–2.0). In patients who did not receive adjuvant 5-FU chemotherapy, CIMP status was the only independent predictor of survival (HR=2.0; 95% CI, 1.1–3.8)
Patients with CIMP+ colorectal tumors do not benefit from 5-FU–based adjuvant chemotherapy.
Colon cancer; 5-FU adjuvant chemotherapy; DNA methylation; response to cancer therapy
KIBRA SNP rs17070145 was identified in a GWAS of memory performance, with some but not all follow-up studies confirming association of its T allele with enhanced memory. This allele was associated with reduced Alzheimer's disease (AD) risk in one study, which also found overexpression of KIBRA in memory-related brain regions of ADs. We genotyped rs17070145 and 14 additional SNPs in 2571 LOADs vs. 2842 controls, including African-Americans. We found significantly reduced risk for rs17070145 T allele in the older African-American subjects (p=0.007) and a suggestive effect in the older Caucasian series. Meta-analysis of this allele in >8000 subjects from our and published series showed a suggestive protective effect (p=0.07). Analysis of episodic memory in control subjects did not identify associations with rs17070145, though other SNPs showed significant associations in one series. KIBRA showed evidence of overexpression in the AD temporal cortex (p=0.06) but not cerebellum. These results suggest a modest role for KIBRA as a cognition and AD risk gene, and also highlight the multifactorial complexity of its genetic associations.
Alzheimer's disease; Association studies in genetics; Case control studies