Thyroid cancer shows high heritability but causative genes remain largely unknown. According to a common hypothesis the genetic predisposition to thyroid cancer is highly heterogeneous; being in part due to many different rare alleles. Here we used linkage analysis and targeted deep sequencing to detect a novel single-nucleotide mutation in chromosome 4q32 (4q32A>C) in a large pedigree displaying non-medullary thyroid carcinoma (NMTC). This mutation is generally ultra-rare; it was not found in 38 NMTC families, in 2676 sporadic NMTC cases or 2470 controls. The mutation is located in a long-range enhancer element whose ability to bind the transcription factors POU2F and YY1 is significantly impaired, with decreased activity in the presence of the C- allele compared with the wild type A-allele. An enhancer RNA (eRNA) is transcribed in thyroid tissue from this region and is greatly downregulated in NMTC tumors. We suggest that this is an example of an ultra-rare mutation predisposing to thyroid cancer with high penetrance.
In this work, we identified a high affinity and potency metallocene-containing triazole peptide conjugate that suppresses the interactions of HIV-1 envelope gp120 at both its CD4 and co-receptor binding sites. The ferrocene-peptide conjugate, HNG-156, was formed by an on-resin copper-catalysed [2 + 3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl-containing triazole conjugate HNG-105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV-1 gp120 due mainly to the decreased dissociation rate of the conjugate-gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037-08) and clade B (YU-2 and SF162) virus subtypes with nanomolar KD in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV-1YU-2 gp120 at both the CD4 binding site and CD4-induced binding sites. HNG-156 showed a close-to nanomolar IC50 for inhibiting cell infection by HIV-1BaL whole virus. The dual receptor site antagonist activity and potency of HNG-156 make it a promising viral envelope inhibitor lead for developing anti-HIV-1 treatments.
HIV-1 gp120; entry inhibitors; peptide triazoles; surface plasmon resonance; cell infection
Mutations in the mismatch repair genes cause Lynch syndrome (LS), conferring high risk of colorectal, endometrial and some other cancers. After the same splice site mutation in the MLH1 gene (c.589-2A>G) had been observed in 4 ostensibly unrelated American families with typical LS cancers, its occurrence in comprehensive series of LS cases (Mayo Clinic, Germany and Italy) was determined. It occurred in 10 out of 995 LS mutation carriers (1.0%) diagnosed in the Mayo Clinic diagnostic laboratory. It did not occur among 1803 cases tested for MLH1 mutations by the German HNPCC consortium, while it occurred in 3 probands and an additional 5 family members diagnosed in Italy. In the U.S., the splice site mutation occurs on a large (~4.8 Mb) shared haplotype that also harbors the variant c.2146G>A which predicts a missense change in codon 716 referred to here as V716M. In Italy, it occurs on a different, shorter shared haplotype (~2.2 Mb) that does not carry V716M. The V716M variant was found to be present by itself in the U.S., German and Italian populations with individuals sharing a common haplotype of 280 kb, allowing us to calculate that the variant arose around 5600 years ago (225 generations; 95% confidence interval 183–272). The splice site mutation in America arose or was introduced some 450 years ago (18 generations; 95% confidence interval 14–23); it accounts for 1.0% all LS in the Unites States and can be readily screened for.
The presence of osmotic gradients in the development of cerebral edema and the effectiveness of osmotherapy are well recognized. A modification of ventriculostomy catheters described in this paper provides a method of osmotherapy that is not currently available. The Reductive Ventricular Osmotherapy (RVOT) catheter removes free water from ventricular cerebrospinal fluid (CSF) by incorporating hollow fibers that remove water vapor, thereby providing osmotherapy without increasing osmotic load.
To increase osmolarity in the ventricular CSF through use of RVOT in vivo.
Twelve Yorkshire swine with contusional injury were randomized to external ventricular drainage (EVD) or RVOT for 12 hours. Magnetic resonance imaging was obtained. Serum, CSF, and brain ultrafiltrate were analyzed. Histology was compared using Fluor-Jade B and H & E.
With RVOT, CSF osmolality increased from 292 ± 2.7 to 345 ± 8.0 mosmol/kg (mean ± SE, p=0.0006), and the apparent diffusion coefficient (ADC) in the injury region increased from 0.735 ±0 .047 to 1.135 ± .063 (p=0.004) over 24 hours. With EVD controls, CSF osmolarity and ADC were not significantly changed. Histologically, all RVOT pigs showed no evidence of neuronal degeneration (Grade 1/4) compared to moderate degeneration (Grade 2.6 +.4/4) seen in EVD treated animals (p=0.02). The difference in intracranial pressure (ICP) by area under the curve approached significance at p = .065 by Mann Whitney test.
RVOT can increase CSF osmolarity in vivo after experimental traumatic brain injury (TBI). In anticipated clinical use, only a slight increase in CSF osmolarity may be required to reduce cerebral edema.
Osmotherapy; Hollow Fibers; Cerebral Edema; Reductive Ventricular Osmotherapy; Porcine; Traumatic Brain Injury
MicroRNA (miR) expression signatures are proposed to be able to differentiate thyroid cancer from benign thyroid lesions. We selected eight miRs (miR-146b, -221, -187, -197, -346, -30d, -138, and -302c) to examine the potential use of miRs to supplement diagnostic cytology in cases designated as “atypia of undetermined significance.”
: miR expression was measured in thyroid fine needle aspiration (FNA) specimens by quantitative polymerase chain reaction. Gene expression analyses and linear discriminant analysis (LDA) were performed in a training sample set (n=60) to obtain a classification rule to predict FNA cases as benign or malignant. The predictions were cross-validated by comparing with the corresponding histological diagnoses. A validation sample set (n=68) was further tested with the established four-miR LDA classification rule.
A set of four miRs (miR-146b, -221, -187, and -30d) was identified that could differentiate malignant from benign lesions. A four-miR LDA classification rule was obtained and used to predict FNA cases as benign or malignant. For the training sample set, we obtained a diagnostic accuracy of 93.3%, sensitivity of 93.2%, specificity of 93.8%, positive predictive value (PPV) of 0.98, and negative predictive value (NPV) of 0.83. For the validation sample set, we obtained a diagnostic accuracy of 85.3%, sensitivity of 88.9%, specificity of 78.3%, PPV of 0.89, and NPV of 0.78. For the 30 atypia cases in the validation sample set, we obtained a diagnostic accuracy of 73.3%, sensitivity of 63.6%, specificity of 78.9%, PPV of 0.64, and NPV of 0.79. Based on the miR predictions, we classified the atypia cases predicted as “malignant” into “high risk” and those predicted as “benign” into “low risk” categories. While thyroid carcinomas, particularly papillary thyroid carcinomas (PTCs), were relatively enriched in the high-risk category, this particular miR panel is subject to inaccurate results in follicular neoplasias in atypia cases.
We demonstrate that miR amplification from FNA samples is feasible and that the particular four miR profile in this study can identify PTCs. However, further refinement is required for application to FNA cytology of “atypia of undetermined significance” cases due to low accuracy in classifying follicular neoplasias.
Background & Aims
Expression of the netrin-1 dependence receptor UNC5C is reduced in many colorectal tumors; mice with the UNC5C mutations have increased progression of intestinal tumors. We investigated whether specific variants in UNC5C increase risk for colorectal cancer (CRC).
We analyzed the sequence of UNC5C in blood samples from 1801 patients with CRC and 4152 controls from 3 cohorts (France, USA, and Finland). Almost all cases from France and the USA had familial CRC; of the Finnish cases, 92/984 were familial. We analyzed whether CRC segregates with the UNC5C variant A628K in 3 families with histories of CRC. We also performed haplotype analysis, to determine the origin of this variant.
Of 817 patients with familial CRC, 14 had 1 of 4 different, unreported missense variants in UNC5C. The variants p.Asp353Asn (encodes D353N), p.Arg603Cys (encodes R603C), and p.Gln630Glu (encodes Q630E) did not occur significantly more often in cases than controls. The variant p.Ala628Lys (A628K) was detected in 3 families in the French cohort (odds ratio [OR], 8.8; Wald’s 95% confidence interval [CI], 1.47–52.93; P=.03) and in 2 families the US cohort (OR, 1.9; P=.6), but was not detected in the Finnish cohort; UNC5C A628K segregated with CRC in families. Three families with A628K had a 109 kb identical haplotype that spanned most of UNC5C, indicating recent origin of this variant in Caucasians (14 generations; 95% CI, 6–36 generations). Transfection of HEK293T cells with UNC5C-A628K significantly reduced apoptosis compared to wild-type UNC5C, measured in an assay of active caspase-3.
Inherited mutations in UNC5C prevent apoptosis and increase risk for CRC.
Colon cancer; tumor suppression; tumorigenesis; neoplasm; UNC5H3
Tuberculosis (TB) is a disease of poverty that contributes significantly to ill-health in developing countries. Drug resistant TB is a major challenge to disease control. Early diagnosis and rapid determination of drug sensitivity is of paramount importance in eradication of TB. Although automated liquid culture based methods are available for rapid detection of drug resistance, the high cost of these tests prevent them from being used routinely in low resource settings. This study compares two phenotypic methods, the manual Mycobacteria Growth Indicator Tube (MGIT) and the Nitrate Reductase Assay (NRA) in liquid medium, with the agar proportion method (APM), the gold standard for susceptibility testing of Mycobacterium tuberculosis.
Fourteen day old M. tuberculosis strains (n=373) grown on solid media were used for drug susceptibility testing by APM, NRA and the manual MGIT method. Rifampicin free and rifampicin incorporated (final concentration, 1 μg/ml) media were inoculated with the recommended concentrations of mycobacterial suspensions and incubated at 37°C in 5% CO2. In the APM, the proportion of colonies in the drug containing medium was determined. In the NRA, the colour change in the medium was compared with a standard colour series after day 6 and day 12 of incubation. Growth in the MGIT was detected using the manual MGIT reader from day 2 onwards. The 2 methods were compared with the gold standard, APM to determine sensitivity and specificity and agreement between the methods was calculated using kappa statistics.
Thirty one (31) rifampicin resistant isolates were identified. When compared with the APM, the sensitivity of detection of rifampicin resistance was 85% for the NRA and 93% for the manual MGIT and the specificity was 99% and 100% respectively. Both assays, NRA (κ=0.86) and manual MGIT method (κ= 0.94) were in excellent agreement with the APM. The mean turnaround time for manual MGIT method and NRA were 08 days and 10 days respectively.
The NRA in liquid medium and manual MGIT are useful alternatives to APM for drug susceptibility testing of M. tuberculosis in low resource settings.
Drug resistant tuberculosis; Determination of drug sensitivity; Manual MGIT; NRA in liquid medium; Agar proportion method
The goal of the Papillomavirus Episteme (PaVE) is to provide an integrated resource for the analysis of papillomavirus (PV) genome sequences and related information. The PaVE is a freely accessible, web-based tool (http://pave.niaid.nih.gov) created around a relational database, which enables storage, analysis and exchange of sequence information. From a design perspective, the PaVE adopts an Open Source software approach and stresses the integration and reuse of existing tools. Reference PV genome sequences have been extracted from publicly available databases and reannotated using a custom-created tool. To date, the PaVE contains 241 annotated PV genomes, 2245 genes and regions, 2004 protein sequences and 47 protein structures, which users can explore, analyze or download. The PaVE provides scientists with the data and tools needed to accelerate scientific progress for the study and treatment of diseases caused by PVs.
Inherited malabsorption of cobalamin (Cbl) causes hematological and neurological abnormalities that can be fatal. Three genes have been implicated in Cbl malabsorption; yet, only about 10% of ~400-500 reported cases have been molecularly studied to date. Recessive mutations in CUBN or AMN cause Imerslund-Gräsbeck Syndrome (IGS), while recessive mutations in GIF cause Intrinsic Factor Deficiency (IFD). IGS and IFD differ in that IGS usually presents with proteinuria, which is not observed in IFD. The genetic heterogeneity and numerous differential diagnoses make clinical assessment difficult.
We present a large genetic screening study of 154 families or patients with suspected hereditary Cbl malabsorption. Patients and their families have been accrued over a period spanning >12 years. Systematic genetic testing of the three genes CUBN, AMN, and GIF was accomplished using a combination of single strand conformation polymorphism and DNA and RNA sequencing. In addition, six genes that were contenders for a role in inherited Cbl malabsorption were studied in a subset of these patients.
Our results revealed population-specific mutations, mutational hotspots, and functionally distinct regions in the three causal genes. We identified mutations in 126/154 unrelated cases (82%). Fifty-three of 126 cases (42%) were mutated in CUBN, 45/126 (36%) were mutated in AMN, and 28/126 (22%) had mutations in GIF. We found 26 undescribed mutations in CUBN, 19 in AMN, and 7 in GIF for a total of 52 novel defects described herein. We excluded six other candidate genes as culprits and concluded that additional genes might be involved.
Cbl malabsorption is found worldwide and genetically complex. However, our results indicate that population-specific founder mutations are quite common. Consequently, targeted genetic testing has become feasible if ethnic ancestry is considered. These results will facilitate clinical and molecular genetic testing of Cbl malabsorption. Early diagnosis improves the lifelong care required by these patients and prevents potential neurological long-term complications. This study provides the first comprehensive overview of the genetics that underlies the inherited Cbl malabsorption phenotype.
Vitamin B12; Cobalamin; Hereditary cobalamin malabsorption; Amnionless; Gastric intrinsic factor; Cubilin; Ancestry; Genetic testing; Founder mutation; Genetic heterogeneity
Alternative NF-κB signaling modulates the activity of PGC-1β to promote oxidative metabolism in skeletal muscle.
Although the physiological basis of canonical or classical IκB kinase β (IKKβ)–nuclear factor κB (NF-κB) signaling pathway is well established, how alternative NF-κB signaling functions beyond its role in lymphoid development remains unclear. In particular, alternative NF-κB signaling has been linked with cellular metabolism, but this relationship is poorly understood. In this study, we show that mice deleted for the alternative NF-κB components IKKα or RelB have reduced mitochondrial content and function. Conversely, expressing alternative, but not classical, NF-κB pathway components in skeletal muscle stimulates mitochondrial biogenesis and specifies slow twitch fibers, suggesting that oxidative metabolism in muscle is selectively controlled by the alternative pathway. The alternative NF-κB pathway mediates this specificity by direct transcriptional activation of the mitochondrial regulator PPAR-γ coactivator 1β (PGC-1β) but not PGC-1α. Regulation of PGC-1β by IKKα/RelB also is mammalian target of rapamycin (mTOR) dependent, highlighting a cross talk between mTOR and NF-κB in muscle metabolism. Together, these data provide insight on PGC-1β regulation during skeletal myogenesis and reveal a unique function of alternative NF-κB signaling in promoting an oxidative metabolic phenotype.
Glioblastoma multiforme (GBM), the most common and aggressive primary brain malignancy, is incurable despite the best combination of current cancer therapies. For the development of more effective therapies, discovery of novel candidate tumor drivers is urgently needed. Here, we report that peroxiredoxin 4 (PRDX4) is a putative tumor driver. PRDX4 levels were highly increased in a majority of human GBMs as well as in a mouse model of GBM. Reducing PRDX4 expression significantly decreased GBM cell growth and radiation resistance in vitro with increased levels of ROS, DNA damage, and apoptosis. In a syngenic orthotopic transplantation model, Prdx4 knockdown limited GBM infiltration and significantly prolonged mouse survival. These data suggest that PRDX4 can be a novel target for GBM therapies in the future.
Small nuclear RNAs (snRNAs) are essential factors in mRNA splicing. By homozygosity mapping and deep sequencing, we show that a gene encoding U4atac snRNA, a component of the minor U12-dependent spliceosome, is mutated in individuals with microcephalic osteodysplastic primordial dwarfism type I (MOPD I), a severe developmental disorder characterized by extreme intrauterine growth retardation and multiple organ abnormalities. Functional assays show that mutations (30G>A, 51G>A, 55G>A, and 111G>A) associated with MOPD I cause defective U12-dependent splicing. Endogenous U12-dependent but not U2-dependent introns are poorly spliced in MOPD I patient fibroblast cells while introduction of wild type U4atac snRNA into MOPD I cells enhances U12-dependent splicing. These results illustrate the critical role of minor intron splicing in human development.
microcephalic osteodysplastic primordial dwarfism type I; RNU4ATAC; mutation; splicing; snRNA; minor spliceosome
Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we demonstrate that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future.
Epigenetic silencing; H3K27me3; DNA methylation; PI3K/AKT signaling; breast cancer
The purpose was to prospectively determine the sensitivity of 64-slice MDCT in detecting and diagnosing the cause of obscure gastrointestinal bleed (OGIB).
Materials and Methods:
Our study included 50 patients (male 30, female 20) in the age range of 3–82 years (average age: 58.52 years) who were referred to our radiology department as part of their workup for clinically evident gastrointestinal (GI) bleed or as part of workup for anemia (with and without positive fecal occult blood test). All patients underwent conventional upper endoscopy and colonoscopy before undergoing CT scan. Following a noncontrast scan, all patients underwent triple-phase contrast CT scan using a 64-slice CT scan system. The diagnostic performance of 64-slice MDCT was compared to the results of capsule endoscopy, 99m-technetium-labeled red blood cell scintigraphy (99mTc-RBC scintigraphy), digital subtraction angiography, and surgery whenever available.
CT scan showed positive findings in 32 of 50 patients. The sensitivity, specificity, positive predictive value, and negative predictive values of MDCT for detection of bleed were 72.2%, 42.8%, 81.2%, and 44.4%, respectively. Capsule endoscopy was done in 15 patients and was positive in 10 patients; it had a sensitivity of 71.4%. Eleven patients had undergone 99mTc-RBC scintigraphy prior to CT scan, and the result was positive in seven patients (sensitivity 70%). Digital subtraction angiography was performed in only eight patients and among them all except one patient showed findings consistent with the lesions detected on MDCT.
MDCT is a sensitive and noninvasive tool that allows rapid detection and localization of OGIB. It can be used as the first-line investigation in patients with negative endoscopy and colonoscopy studies. MDCT and capsule endoscopy have complementary roles in the evaluation of OGIB.
Arteriography digital subtraction; capsule endoscopy; diagnosis; gastrointestinal hemorrhage; spiral cone-beam computed tomography
Imerslund-Gräsbeck syndrome (IGS) was described just over 50 years ago by Olga Imerslund and Ralph Gräsbeck and colleagues. IGS is caused by specific malabsorption of cobalamin (Cbl) due to bi-allelic mutations in either the cubilin gene (CUBN) or the human amnionless homolog (AMN). Mutations in the two genes are commonly seen in founder populations or in societies with a high degree of consanguineous marriages. One particular mutation in AMN, c.208-2A>G, causing an out-of-frame loss of exon 4 in the mRNA, is responsible for some 15% of IGS cases globally. We present evidence that this founder mutation causes a substantial percentage of cases among diverse ethnicities and that the mutation is as old as human civilization.
Partial genotyping indicated a founder event but its presence in diverse peoples of Arabic, Turkish, Jewish, and Hispanic ancestry suggested that the mutation might be recurrent. We therefore studied the flanking sequence spanning 3.5 Mb to elucidate the origin of the haplotype and estimate the age of the mutation using a Bayesian inference method based on observed linkage disequilibrium.
The mutation's distribution, the size of the shared haplotype, and estimates of growth rate and carrier frequency indicated that the mutation was a single prehistoric event. Dating back to the ancient Middle East around 11,600 BC, the mutation predates the advent of writing, farming, and the monotheistic religions of the region.
This mutation causes over 50% of the IGS cases among Arabic, Turkish, and Sephardic Jewish families, making it a primary target for genetic screening among diverse IGS cases originating from the Middle East. Thus, rare founder mutations may cause a substantial number of cases, even among diverse ethnicities not usually thought to be related.
Imerslund-Gräsbeck syndrome; juvenile cobalamin deficiency; founder mutation; age estimation; mutation screening, anemia; ethnicity
The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.
Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. The aim of this study was to examine epigenetic changes in breast epithelial cells treated with low-dose BPA. We also investigated the effect of BPA on the ERα signaling pathway and global gene expression profiles. Compared to control cells, nuclear internalization of ERα was observed in epithelial cells preexposed to BPA. We identified 170 genes with similar expression changes in response to BPA. Functional analysis confirms that gene suppression was mediated in part through an ERα-dependent pathway. As a result of exposure to BPA or other estrogen-like chemicals, the expression of lysosomal-associated membrane protein 3 (LAMP3) became epigenetically silenced in breast epithelial cells. Furthermore, increased DNA methylation in the LAMP3 CpG island was this repressive mark preferentially occurred in ERα-positive breast tumors. These results suggest that the in vitro system developed in our laboratory is a valuable tool for exposure studies of BPA and other xenoestrogens in human cells. Individual and geographical differences may contribute to altered patterns of gene expression and DNA methylation in susceptible loci. Combination of our exposure model with epigenetic analysis and other biochemical assays can give insight into the heritable effect of low-dose BPA in human cells.
Bisphenol A; Estrogen; DNA methylation; Epigenetics; Breast cancer
A hallmark of malignant gliomas is their ability to disperse through neural tissue, leading to long-term failure of all known therapies. Identifying new antimigratory targets could reduce glioma recurrence and improve therapeutic efficacy, but screens based on conventional migration assays are hampered by the limited ability of these assays to reproduce native cell motility. Here, we have analyzed the motility, gene expression, and sensitivity to migration inhibitors of glioma cells cultured on scaffolds formed by submicron-sized fibers (nanofibers) mimicking the neural topography. Glioma cells cultured on aligned nanofiber scaffolds reproduced the elongated morphology of cells migrating in white matter tissue and were highly sensitive to myosin II inhibition but only moderately affected by stress fiber disruption. In contrast, the same cells displayed a flat morphology and opposite sensitivity to myosin II and actin inhibition when cultured on conventional tissue culture polystyrene. Gene expression analysis indicated a correlation between migration on aligned nanofibers and increased STAT3 signaling, a known driver of glioma progression. Accordingly, cell migration out of glioblastoma-derived neurospheres and tumor explants was reduced by STAT3 inhibitors at subtoxic concentrations. Remarkably, these inhibitors were ineffective when tested at the same concentrations in a conventional two-dimensional migration assay. We conclude that migration of glioma cells is regulated by topographical cues that affect cell adhesion and gene expression. Cell migration analysis using nanofiber scaffolds could be used to reproduce native mechanisms of migration and to identify antimigratory strategies not disclosed by other in vitro models.
Aurora B activity is inhibited when centromeric repeat sequences are absent, although kinetochores can still assemble.
The nearly ubiquitous presence of repetitive centromere DNA sequences across eukaryotic species is in paradoxical contrast to their apparent functional dispensability. Centromeric chromatin is spatially delineated into the kinetochore-forming array of centromere protein A (CENP-A)–containing nucleosomes and the inner centromeric heterochromatin that lacks CENP-A but recruits the aurora B kinase that is necessary for correcting erroneous attachments to the mitotic spindle. We found that the self-perpetuating network of CENPs at the foundation of the kinetochore is intact at a human neocentromere lacking repetitive α-satellite DNA. However, aurora B is inappropriately silenced as a consequence of the altered geometry of the neocentromere, thereby compromising the error correction mechanism. This suggests a model wherein the neocentromere represents a primordial inheritance locus that requires subsequent generation of a robust inner centromere compartment to enhance fidelity of chromosome transmission.
The genetic component of colorectal cancer (CRC) predisposition has been only partially explained. We recently suggested that a subtle decrease in the expression of one allele of the TGFBR1 gene was a heritable quantitative trait predisposing to CRC. Here, we refined the measurements of allele-specific expression (ASE) of TGFBR1 in a population-based series of CRC patients and controls. Five single-nucleotide polymorphisms (SNPs) in the 3′-untranslated region of the gene were genotyped and used for ASE determination by pyrosequencing. After eliminating non-informative samples and samples with RNA of insufficient quality 109 cases and 125 controls were studied. Allelic ratios ranged between 0.74 and 1.69 without evidence of bimodality or cutoff points for ‘ASE’ versus ‘non-ASE’. Treating ASE as a continuous variable, cases had non-significantly different values than controls (P = 0.081 when comparing means by permutation test). However, cases had significantly higher ASE values when comparing medians by permutation test (P = 0.0027) and when using Wilcoxon test (P = 0.0094). We conclude that with the present-day technology, ASE differences between individuals and between cases and controls are too subtle to be used to assess CRC risk. More advanced technology is expected to resolve this issue as well as the low informativity caused by the limited heterozygosity of transcribed SNPs.
Early exposure to xenoestrogens may predispose to breast cancer risk later in adult life. It is likely that long-lived, self-regenerating epithelial progenitor cells are more susceptible to these exposure injuries over time and transmit the injured memory through epigenetic mechanisms to their differentiated progeny. Here, we used progenitor-containing mammospheres as an in vitro exposure model to study this epigenetic effect. Expression profiling identified that, relative to control cells, 9.1% of microRNAs (82 of 898 loci) were altered in epithelial progeny derived from mammospheres exposed to a synthetic estrogen, diethylstilbestrol. Repressive chromatin marks, trimethyl Lys27 of histone H3 (H3K27me3) and dimethyl Lys9 of histone H3 (H3K9me2), were found at a down-regulated locus, miR-9-3, in epithelial cells preexposed to diethylstilbestrol. This was accompanied by recruitment of DNA methyltransferase 1 that caused an aberrant increase in DNA methylation of its promoter CpG island in mammosphere-derived epithelial cells on diethylstilbestrol preexposure. Functional analyses suggest that miR-9-3 plays a role in the p53-related apoptotic pathway. Epigenetic silencing of this gene, therefore, reduces this cellular function and promotes the proliferation of breast cancer cells. Promoter hypermethylation of this microRNA may be a hallmark for early breast cancer development, and restoration of its expression by epigenetic and microRNA-based therapies is another viable option for future treatment of this disease.
Rationale: Little is known about the genetic regulation of granulomatous inflammation in sarcoidosis.
Objectives: To determine if tissue gene array analysis would identify novel genes engaged in inflammation and lung remodeling in patients with sarcoidosis.
Methods: Gene expression analysis was performed on tissues obtained from patients with sarcoidosis at the time of diagnosis (untreated) (n = 6) compared with normal lung tissue (n = 6). Expression of select genes was further confirmed in lung tissue from a second series of patients with sarcoidosis and disease-free control subjects (n = 11 per group) by semi-quantitative RT-PCR. Interactive gene networks were identified in patients with sarcoidosis using Ingenuity Pathway Analysis (Ingenuity Systems, Inc., Redwood, CA) software. The expression of proteins corresponding to selected overexpressed genes was determined using fluorokine multiplex analysis, and immunohistochemistry. Selected genes and proteins were then analyzed in bronchoalveolar lavage fluid in an independent series of patients with sarcoidosis (n = 36) and control subjects (n = 12).
Measurements and Main Results: A gene network engaged in Th1-type responses was most significantly overexpressed in the sarcoidosis lung tissues, including genes not previously reported in the context of sarcoidosis (e.g., IL-7). MMP-12 and ADAMDEC1 transcripts were most highly expressed (> 25-fold) in sarcoidosis lung tissues, corresponding with increased protein expression by immunohistochemistry. MMP-12 and ADAMDEC1 gene and protein expression were increased in bronchoalveolar lavage samples from patients with sarcoidosis, correlating with disease severity.
Conclusions: Tissue gene expression analyses provide novel insights into the pathogenesis of pulmonary sarcoidosis. MMP-12 and ADAMDEC1 emerge as likely mediators of lung damage and/or remodeling and may serve as markers of disease activity.
genetic; gene array; granuloma; lung; BAL
Ovarian cancer ranks the most lethal among gynecologic neoplasms in women. To develop potential bio-markers for diagnosis, we have identified five novel genes (CYP39A1, GTF2A1, FOXD4L4, EBP, and HAAO) that are hypermethylated in ovarian tumors, compared with the non-malignant normal ovarian surface epithelia, using the quantitative methylation-specific polymerase chain reactions. Interestingly enough, multivariate Cox regression analysis has identified hypermethylation of CYP39A1 correlated with an increase rate of relapsing (P=0.032, hazard ratio >1). Concordant hypermethylation in at least three loci was observed in 50 out of 55 (91%) of ovarian tumors examined. The test sensitivity and specificity were assessed to be 96 and 67% for CYP39A1; 95 and 88% for GTF2A1; 93 and 67% for FOXD4L4; 81 and 67% for EBP; 89 and 82% for HAAO, respectively. Our data have identified, for the first time, GTF2A1 alone, or GTF2A1 plus HAAO are excellent candidate biomarkers for detecting this disease. Moreover, the known functions of these gene products further implicate dys-regulated transcriptional control, cholesterol metabolism, or synthesis of quinolinic acids, may play important roles in attributing to ovarian neoplasm. Molecular therapies, by reversing the aberrant epigenomes using inhibitory agents or by abrogating the upstream signaling pathways that convey the epigenomic perturbations, may be developed into promising treatment regimens.
ovarian cancer; epigenetics; DNA methylation; biomarkers; quantitative methylation-specific polymerase chain reaction
The interplay between histone modifications and promoter hypermethylation provides a causative explanation for epigenetic gene silencing in cancer. Less is known about the upstream initiators that direct this process. Here, we report that the Cystatin M (CST6) tumor suppressor gene is concurrently down-regulated with other loci in breast epithelial cells co-cultured with cancer-associated fibroblasts (CAFs). Promoter hypermethylation of CST6 is associated with aberrant AKT1 activation in epithelial cells, as well as the disabled INNP4B regulator resulted from the suppression by CAFs. Repressive chromatin, marked by trimethyl-H3K27 and dimethyl-H3K9, and de novo DNA methylation is established at the promoter. The findings suggest that microenvironmental stimuli are triggers in this epigenetic cascade, leading to the long-term silencing of CST6 in breast tumors. Our present findings implicate a causal mechanism defining how tumor stromal fibroblasts support neoplastic progression by manipulating the epigenome of mammary epithelial cells. The result also highlights the importance of direct cell-cell contract between epithelial cells and the surrounding fibroblasts that confer this epigenetic perturbation. Since this two-way interaction is anticipated, the described co-culture system can be used to determine the effect of epithelial factors on fibroblasts in future studies.