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Routine use of completion imaging after infrainguinal bypass is not associated with higher bypass graft patency

Tue, 12/30/2014 - 10:57am

BACKGROUND: Significant variability exists in completion imaging (CIM) after infrainguinal lower extremity bypass (LEB). We evaluated the use of CIM and compared graft patency in patients treated by surgeons who performed routine CIM vs those who performed selective CIM.

METHODS: We reviewed the Vascular Study Group of New England database (2003-2010) and assessed the use of CIM (angiography or duplex ultrasound) among patients undergoing LEB. The surgeon-specific CIM strategy was categorized as routine ( > / = 80% of LEBs) vs selective ( < 80% of LEBs). Exclusion criteria included acute limb ischemia, bilateral procedures, and surgeon volume < 10 cases per study period. Primary graft patency at discharge and at 1 year was analyzed on the basis of CIM use and surgeon-specific CIM strategy. Multivariable analyses were performed using Poisson regression.

RESULTS: Among 2032 LEB procedures performed by 48 surgeons, CIM was used in 1368 cases (67.3%). CIM was performed in 72% of autogenous LEBs and 52% of prosthetic grafts. Dialysis (odds ratio [OR], 1.7; 95% confidence interval [CI], 1.1-2.6; P = .01), elective LEB (OR, 2.6; 95% CI, 1.4-4.8; P = .002), great saphenous vein conduit (OR, 2.0; 95% CI, 1.6-2.5; P < .001), and tibial or pedal target artery (OR, 1.8; 95% CI, 1.4-2.3; P < .001) were associated with CIM use. In multivariate models, CIM was not associated with improved primary graft patency at discharge (OR, 1.1; 95% CI, 0.7-1.7; P = .64) or at 1 year (OR, 0.9; 95% CI, 0.7-1.2; P = .47). Sixteen surgeons (33%) were routine users and 32 (67%) were selective users of CIM. Among patients of routine vs selective CIM users, primary graft patency at discharge and at 1 year was 96% vs 94% (P = .21) and 68% vs 72% (P = .09), respectively. In multivariate analysis, routine or selective CIM strategy was not associated with improved discharge (rate ratio, 0.8; 95% CI, 0.6-1.1; P = .31) or 1-year (rate ratio, 1.1; 95% CI, 0.9-1.2; P = .56) graft patency.

CONCLUSIONS: In our observational cohort, CIM does not improve short-term and 1-year bypass graft patency in infrainguinal LEB. The surgeon-specific strategy of selective CIM after LEB has outcomes comparable to those of routine CIM. Copyright © 2014 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.

Factors that determine the length of stay after carotid endarterectomy represent opportunities to avoid financial losses

Tue, 12/30/2014 - 10:57am

BACKGROUND: A postoperative length of stay (LOS) > 1 day after elective surgery incurs financial losses for hospitals, given fixed diagnosis-related group-based reimbursement. We sought to identify factors leading to a prolonged LOS ( > 1 postoperative day) after carotid endarterectomy (CEA). METHODS: Patients undergoing CEA in 23 centers of the Vascular Study Group of New England between 2003 and 2011 (n = 8860) were analyzed. Only elective, primary CEAs were analyzed, leaving a study cohort of 7108 procedures. Hierarchical multivariable logistic regression analysis was performed to identify predictors of a postoperative LOS > 1 day. A Knaus-Wagner chi-pie analysis was performed to determine the relative contributions of each significant covariate to a postoperative LOS > 1 day. RESULTS: A postoperative LOS > 1 day occurred in 17.5% of the sample (n = 1244). The average LOS was 1.4 days (range, 1-91 days; median, 1). There was significant variation in rates of postoperative LOS > 1 day across centers (range, 5%-100%; P < .001). Factors independently associated with a postoperative LOS > 1 day and their percentage contribution to the prediction model included the need for postoperative intravenous medications for hypertension or hypotension (26%), any major adverse event (MAE) postoperatively (21%), low-volume ( < 15 CEAs per year) surgeons (28%), increasing age (7%), female gender (4%), positive result on a preoperative stress test (3%), preoperative major stroke < / = 30 days (2%), medication-dependent diabetes (1%), severe chronic obstructive pulmonary disease (1%), history of congestive heart failure (1%), and CEA performed on Friday (2%). CONCLUSIONS: Certain patient characteristics predispose to a postoperative LOS > 1 day after elective CEA. However, patient characteristics play only a modest (17%) role in determining LOS. The need for postoperative blood pressure control and MAEs are the biggest drivers of postoperative LOS > 1 day, but system factors, such as low operative volume, contribute substantially to postoperative LOS > 1 day, independent of MAEs. These findings can be used to guide quality improvement efforts designed to reduce LOS after elective CEA. rights reserved.

Outcomes of carotid endarterectomy versus stenting in comparable medical risk patients

Tue, 12/30/2014 - 10:57am

OBJECTIVE: In medically high-risk patients the choice between carotid artery stenting (CAS) and carotid endarterectomy (CEA) can be difficult. The purpose of this study was to compare risk-stratified outcomes of CAS and CEA. METHODS: Patients who underwent isolated primary CEA (n = 11,336) or primary CAS (n = 544) at 29 centers in the Vascular Study Group of New England were analyzed (2003-2013); patients with previous ipsilateral CEA or CAS, or concomitant coronary artery bypass graft were excluded. A medical risk score based on predicted 5-year mortality was developed for each patient using a Cox proportional hazards model. Patients in the highest risk score quartile were termed high-risk (vs normal-risk for the other three quartiles). Medically high-risk patients had a 5-year survival of 65% and comprised 23% of CEA and 25% of CAS patients. Risk-stratified outcomes were compared within neurologically symptomatic and asymptomatic patients. RESULTS: Among asymptomatic patients, rates of in-hospital stroke and/or death were not different between CAS and CEA in normal and high-risk cohorts, ranging from 0.7% in normal-risk CEA patients to 1.6% in high-risk CAS patients. In symptomatic patients, significantly worse outcomes were seen with CAS compared with CEA in normal-risk and high-risk patients. Normal-risk symptomatic patients had a stroke or death rate of 1.3% with CEA, but 5.2% with CAS (P < .01). In high-risk symptomatic patients, the stroke or death rate was 1.5% with CEA and 9.3% with CAS (P < .01). No significant differences were seen between asymptomatic CEA and CAS within risk strata across secondary outcome measures of stroke, death, or myocardial infarction, and ipsilateral stroke, major stroke, or death. However, symptomatic high-risk CAS patients had significantly greater rates of all secondary outcomes compared with CEA except death, and symptomatic normal-risk CAS patients had only significantly greater rates of death and stroke, death, or myocardial infarction. CONCLUSIONS: In the Vascular Study Group of New England, asymptomatic normal- and high-risk patients do equally well after CEA or CAS. However, normal- and high-risk symptomatic patients have substantially worse outcomes with CAS compared with CEA. High medical risk alone might be an insufficient indication for CAS in symptomatic patients. rights reserved.

The influence of gender on functional outcomes of lower extremity bypass

Tue, 12/30/2014 - 10:57am

OBJECTIVE: Our aim was to evaluate the effect of gender on early and late procedural and functional outcomes of lower extremity bypass (LEB). METHODS: We reviewed the records of 2576 patients (828 women; 32%) who underwent LEB for claudication or critical limb ischemia (CLI) in the Vascular Study Group of New England from 2003 to 2010. Logistic regression and proportional hazards models were used to adjust for potential confounding differences between genders. Morbidity, mortality, graft patency, freedom from major amputation, ambulation, and living status were analyzed postoperatively and over 1 year. RESULTS: Women were older (70 vs 68 years; P < .001), had more hypertension (89% vs 85%; P = .006), less coronary artery disease (35% vs 39%; P = .03), smoking (73% vs 88%; P < .001), and preoperative statin use (60% vs 64%; P = .04). Women were more likely to have CLI (76% vs 71%; P = .003), and ambulate with assistance at presentation (19% vs 16%; P = .02). Morbidity was similar except women had higher rates of reoperation for thrombosis (4% vs 2%; P < .001) without differences in major amputation (2% vs 1%; P = .13) or in-hospital mortality (1.7% vs 1.7%; P = .96). Women and men with claudication had similar 1-year graft patency rates. Women with CLI had lower rates of primary (hazard ratio [HR], 1.24; 95% confidence interval [CI], 1.03-1.48; P = .02), assisted primary (HR, 1.42; 95% CI, 1.15-1.76; P = .001) and secondary patency (HR, 1.40; 95% CI, 1.10-1.77; P = .006) during the first year compared with men. Freedom from amputation was similar for men and women with CLI (HR, 1.17; 95% CI, 0.84-1.63; P = .36). There were no differences in late survival between women and men with claudication (HR, 0.89; 95% CI, 0.60-1.31; P = .36) or CLI (HR, 0.94; 95% CI, 0.81-1.09; P = .39). More female claudicants were not independently ambulatory at discharge (30% vs 19%; P = .002) and were discharged to a nursing home (15% vs 5%; P < .001) but these differences did not persist at 1 year. Women with CLI were more likely to be nonambulatory at discharge (13% vs 9%; P = .006) and at 1 year (13% vs 8%; P < .001). More women with CLI were discharged to a nursing home (44% vs 35%; P = .01) and resided there at 1 year (11% vs 7%; P = .02). CONCLUSIONS: Women have complication rates similar to men with inferior early and late functional outcomes after LEB. The reduced patency rates in women with CLI did not translate into differences in limb salvage. These findings might help define physician and patient expectations for women before revascularization. rights reserved.

Follow-up compliance after endovascular abdominal aortic aneurysm repair in Medicare beneficiaries

Tue, 12/30/2014 - 10:57am

OBJECTIVE: Lifelong imaging follow-up is essential to the safe and appropriate management of patients who undergo endovascular abdominal aortic aneurysm repair (EVAR). We sought to evaluate the rate of compliance with imaging follow-up after EVAR and to identify factors associated with being lost to imaging follow-up. METHODS: We identified a 20% sample of continuously enrolled Medicare beneficiaries who underwent EVAR between 2001 and 2008. Using data through 2010 from Medicare Inpatient, Outpatient, and Carrier files, we identified all abdominal imaging studies that may have been performed for EVAR follow-up. Patients were considered lost to annual imaging follow-up if they did not undergo any abdominal imaging study within their last 2 years of follow-up. Multivariable models were constructed to identify independent factors associated with being lost to annual imaging follow-up. RESULTS: Among 19,962 patients who underwent EVAR, the incidence of loss to annual imaging follow-up at 5 years after EVAR was 50%. Primary factors associated with being lost to annual imaging follow-up were advanced age (age 65-69 years, reference; age 75-79 years: hazard ratio [HR], 1.23; 95% confidence interval [CI], 1.15-1.32; age 80-85 years: HR, 1.45; 95% CI, 1.35-1.55; age > 85 years: HR, 2.03; 95% CI, 1.88-2.20) and presentation with an urgent/emergent intact aneurysm (HR, 1.27; 95% CI, 1.20-1.35) or ruptured aneurysm (HR, 1.84; 95% CI, 1.63-2.08). Additional independent factors included several previously diagnosed chronic diseases and South and West regions of the United States. CONCLUSIONS: Annual imaging follow-up compliance after EVAR in the United States is significantly below recommended levels. Quality improvement efforts to encourage improved compliance with imaging follow-up, especially in older patients with multiple comorbidities and in those who underwent EVAR urgently or for rupture, are necessary. rights reserved.

The nucleoporin Seh1 forms a complex with Mio and serves an essential tissue-specific function in Drosophila oogenesis

Mon, 12/22/2014 - 7:30pm

The nuclear pore complex (NPC) mediates the transport of macromolecules between the nucleus and cytoplasm. Recent evidence indicates that structural nucleoporins, the building blocks of the NPC, have a variety of unanticipated cellular functions. Here, we report an unexpected tissue-specific requirement for the structural nucleoporin Seh1 during Drosophila oogenesis. Seh1 is a component of the Nup107-160 complex, the major structural subcomplex of the NPC. We demonstrate that Seh1 associates with the product of the missing oocyte (mio) gene. In Drosophila, mio regulates nuclear architecture and meiotic progression in early ovarian cysts. Like mio, seh1 has a crucial germline function during oogenesis. In both mio and seh1 mutant ovaries, a fraction of oocytes fail to maintain the meiotic cycle and develop as pseudo-nurse cells. Moreover, the accumulation of Mio protein is greatly diminished in the seh1 mutant background. Surprisingly, our characterization of a seh1 null allele indicates that, although required in the female germline, seh1 is dispensable for the development of somatic tissues. Our work represents the first examination of seh1 function within the context of a multicellular organism. In summary, our studies demonstrate that Mio is a novel interacting partner of the conserved nucleoporin Seh1 and add to the growing body of evidence that structural nucleoporins can have novel tissue-specific roles.

Identifying diagnostic DNA methylation profiles for facioscapulohumeral muscular dystrophy in blood and saliva using bisulfite sequencing

Mon, 12/22/2014 - 7:30pm

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is linked to chromatin relaxation due to epigenetic changes at the 4q35 D4Z4 macrosatellite array. Molecular diagnostic criteria for FSHD are complex and involve analysis of high molecular weight (HMW) genomic DNA isolated from lymphocytes, followed by multiple restriction digestions, pulse-field gel electrophoresis (PFGE), and Southern blotting. A subject is genetically diagnosed as FSHD1 if one of the 4q alleles shows a contraction in the D4Z4 array to below 11 repeats, while maintaining at least 1 repeat, and the contraction is in cis with a disease-permissive A-type subtelomere. FSHD2 is contraction-independent and cannot be diagnosed or excluded by this common genetic diagnostic procedure. However, FSHD1 and FSHD2 are linked by epigenetic deregulation, assayed as DNA hypomethylation, of the D4Z4 array on FSHD-permissive alleles. We have developed a PCR-based assay that identifies the epigenetic signature for both types of FSHD, distinguishing FSHD1 from FSHD2, and can be performed on genomic DNA isolated from blood, saliva, or cultured cells.

RESULTS: Samples were obtained from healthy controls or patients clinically diagnosed with FSHD, and include both FSHD1 and FSHD2. The genomic DNAs were subjected to bisulfite sequencing analysis for the distal 4q D4Z4 repeat with an A-type subtelomere and the DUX4 5' promoter region. We compared genomic DNA isolated from saliva and blood from the same individuals and found similar epigenetic signatures. DNA hypomethylation was restricted to the contracted 4qA chromosome in FSHD1 patients while healthy control subjects were hypermethylated. Candidates for FSHD2 showed extreme DNA hypomethylation on the 4qA DUX4 gene body as well as all analyzed DUX4 5' sequences. Importantly, our assay does not amplify the D4Z4 arrays with non-permissive B-type subtelomeres and accurately excludes the arrays with non-permissive A-type subtelomeres.

CONCLUSIONS: We have developed an assay to identify changes in DNA methylation on the pathogenic distal 4q D4Z4 repeat. We show that the DNA methylation profile of saliva reflects FSHD status. This assay can distinguish FSHD from healthy controls, differentiate FSHD1 from FSHD2, does not require HMW genomic DNA or PFGE, and can be performed on either cultured cells, tissue, blood, or saliva samples.

Association of modified cytosines and the methylated DNA-binding protein MeCP2 with distinctive structural domains of lampbrush chromatin

Mon, 12/22/2014 - 7:30pm

We have investigated the association of DNA methylation and proteins interpreting methylation state with the distinctive closed and open chromatin structural domains that are directly observable in the lampbrush chromosomes (LBCs) of amphibian oocytes. To establish the distribution in LBCs of MeCP2, one of the key proteins binding 5-methylcytosine-modified DNA (5mC), we expressed HA-tagged MeCP2 constructs in Xenopus laevis oocytes. Full-length MeCP2 was predominantly targeted to the closed, transcriptionally inactive chromomere domains in a pattern proportional to chromomeric DNA density and consistent with a global role in determining chromatin state. A minor fraction of HA-MeCP2 was also found to associate with a distinctive structural domain, namely a short region at the bases of some of the extended lateral loops. Expression in oocytes of deleted constructs and of point mutants derived from Rett syndrome patients demonstrated that the association of MeCP2 with LBCs was determined by its 5mC-binding domain. We also examined more directly the distribution of 5mC by immunostaining Xenopus and axolotl LBCs and confirmed the pattern suggested by MeCP2 targeting of intense staining of the chromomeres and of some loop bases. In addition, we found in the longer loops of axolotl LBCs that short interstitial regions could also be clearly stained for 5mC. These 5mC regions corresponded precisely to unusual segments of active transcription units from which RNA polymerase II (pol II) and nascent transcripts were simultaneously absent. We also examined by immunostaining the distribution in lampbrush chromatin of the oxidized 5mC derivative, 5-hydroxymethylcytosine (5hmC). Although in general, the pattern resembled that obtained for 5mC, one antibody against 5hmC produced intense staining of restricted chromosomal foci. These foci corresponded to a third type of lampbrush chromatin domain, the transcriptionally active but less extended structures formed by clusters of genes transcribed by pol III. This raises the possibility that 5hmC may play a role in establishing the distinctive patterns of gene repression and activation that characterize specific pol III-transcribed gene families in amphibian genomes.

C. elegans PAT-9 is a nuclear zinc finger protein critical for the assembly of muscle attachments

Mon, 12/22/2014 - 7:30pm

BACKGROUND: Caenorhabditis elegans sarcomeres have been studied extensively utilizing both forward and reverse genetic techniques to provide insight into muscle development and the mechanisms behind muscle contraction. A previous genetic screen investigating early muscle development produced 13 independent mutant genes exhibiting a Pat (paralyzed and arrested elongation at the two-fold length of embryonic development) muscle phenotype. This study reports the identification and characterization of one of those genes, pat-9.

RESULTS: Positional cloning, reverse genetics, and plasmid rescue experiments were used to identify the predicted C. elegans gene T27B1.2 (recently named ztf-19) as the pat-9 gene. Analysis of pat-9 showed it is expressed early in development and within body wall muscle lineages, consistent with a role in muscle development and producing a Pat phenotype. However, unlike most of the other known Pat gene family members, which encode structural components of muscle attachment sites, PAT-9 is an exclusively nuclear protein. Analysis of the predicted PAT-9 amino acid sequence identified one putative nuclear localization domain and three C2H2 zinc finger domains. Both immunocytochemistry and PAT-9::GFP fusion expression confirm that PAT-9 is primarily a nuclear protein and chromatin immunoprecipitation (ChIP) experiments showed that PAT-9 is present on certain gene promoters.

CONCLUSIONS: We have shown that the T27B1.2 gene is pat-9. Considering the Pat-9 mutant phenotype shows severely disrupted muscle attachment sites despite PAT-9 being a nuclear zinc finger protein and not a structural component of muscle attachment sites, we propose that PAT-9 likely functions in the regulation of gene expression for some necessary structural or regulatory component(s) of the muscle attachment sites.

Facioscapulohumeral muscular dystrophy region gene 1 is a dynamic RNA-associated and actin-bundling protein

Mon, 12/22/2014 - 7:30pm

FSHD region gene 1 (FRG1) is a dynamic nuclear and cytoplasmic protein that, in skeletal muscle, shows additional localization to the sarcomere. Maintaining appropriate levels of FRG1 protein is critical for muscular and vascular development in vertebrates; however, its precise molecular function is unknown. This study investigates the molecular functions of human FRG1, along with mouse FRG1 and Xenopus frg1, using molecular, biochemical, and cellular-biological approaches, to provide further insight into its roles in vertebrate development. The nuclear fraction of the endogenous FRG1 is localized in nucleoli, Cajal bodies, and actively transcribed chromatin; however, contrary to overexpressed FRG1, the endogenous FRG1 is not associated with nuclear speckles. We characterize the nuclear and nucleolar import of FRG1, the potential effect of phosphorylation, and its interaction with the importin karyopherin alpha2. Consistent with a role in RNA biogenesis, human FRG1 is associated with mRNA in vivo and invitro, interacts directly with TAP (Tip-associated protein; the major mRNA export receptor), and is a dynamic nuclear-cytoplasmic shuttling protein supporting a function for FRG1 in mRNA transport. Biochemically, we characterize FRG1 actin binding activity and show that the cytoplasmic pool of FRG1 is dependent on an intact actin cytoskeleton for its localization. These data provide the first biochemical activities (actin binding and RNA binding) for human FRG1 and the characterization of the endogenous human FRG1, together indicating that FRG1 is involved in multiple aspects of RNA biogenesis, including mRNA transport and, potentially, cytoplasmic mRNA localization.

Temporal uncoupling of the DNA methylome and transcriptional repression during embryogenesis

Mon, 12/22/2014 - 7:30pm

DNA methylation is a tightly regulated epigenetic mark associated with transcriptional repression. Next-generation sequencing of purified methylated DNA obtained from early Xenopus tropicalis embryos demonstrates that this genome is heavily methylated during blastula and gastrula stages. Although DNA methylation is largely absent from transcriptional start sites marked with histone H3 lysine 4 trimethylation (H3K4me3), we find both promoters and gene bodies of active genes robustly methylated. In contrast, DNA methylation is absent in large H3K27me3 domains, indicating that these two repression pathways have different roles. Comparison with chromatin state maps of human ES cells reveals strong conservation of epigenetic makeup and gene regulation between the two systems. Strikingly, genes that are highly expressed in pluripotent cells and in Xenopus embryos but not in differentiated cells exhibit relatively high DNA methylation. Therefore, we tested the repressive potential of DNA methylation using transient and transgenic approaches and show that methylated promoters are robustly transcribed in blastula- and gastrula-stage embryos, but not in oocytes or late embryos. These findings have implications for reprogramming and the epigenetic regulation of pluripotency and differentiation and suggest a relatively open, pliable chromatin state in early embryos followed by reestablished methylation-dependent transcriptional repression during organogenesis and differentiation.

A brain-derived MeCP2 complex supports a role for MeCP2 in RNA processing

Mon, 12/22/2014 - 7:30pm

Mutations in MECP2 (methyl-CpG-binding protein 2) are linked to the severe postnatal neurodevelopmental disorder RTT (Rett syndrome). MeCP2 was originally characterized as a transcriptional repressor that preferentially bound methylated DNA; however, recent results indicate MeCP2 is a multifunctional protein. MeCP2 binding is now associated with certain expressed genes and involved in nuclear organization as well, indicating that its gene regulatory function is context-dependent. In addition, MeCP2 is proposed to regulate mRNA splicing and a mouse model for RTT shows aberrant mRNA splicing. To further understand MeCP2 and potential roles in RTT pathogenesis, we have employed a biochemical approach to identify the MeCP2 protein complexes present in the mammalian brain. We show that MeCP2 exists in at least four biochemically distinct pools in the brain and characterize one novel brain-derived MeCP2 complex that contains the splicing factor Prpf3 (pre-mRNA processing factor 3). MeCP2 directly interacts with Prpf3 in vitro and in vivo and many MECP2 RTT truncations disrupt the MeCP2-Prpf3 complex. In addition, MeCP2 and Prpf3 associate in vivo with mRNAs from genes known to be expressed when their promoters are associated with MeCP2. These results support a role for MeCP2 in mRNA biogenesis and suggest an additional mechanism for RTT pathophysiology.

Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a dynamic nuclear and sarcomeric protein

Mon, 12/22/2014 - 7:30pm

Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a candidate gene for mediating FSHD pathophysiology, however, very little is known about the endogenous FRG1 protein. This study uses immunocytochemistry (ICC) and histology to provide insight into FRG1's role in vertebrate muscle development and address its potential involvement in FSHD pathophysiology. In cell culture, primary myoblast/myotube cultures, and mouse and human muscle sections, FRG1 showed distinct nuclear and cytoplasmic localizations and nuclear shuttling assays indicated the subcellular pools of FRG1 are linked. During myoblast differentiation, FRG1's subcellular distribution changed dramatically with FRG1 eventually associating with the matured Z-discs. This Z-disc localization was confirmed using isolated mouse myofibers and found to be maintained in adult human skeletal muscle biopsies. Thus, FRG1 is not likely involved in the initial assembly and alignment of the Z-disc but may be involved in sarcomere maintenance or signaling. Further analysis of human tissue showed FRG1 is strongly expressed in arteries, veins, and capillaries, the other prominently affected tissue in FSHD. Overall, we show that in mammalian cells, FRG1 is a dynamic nuclear and cytoplasmic protein, however in muscle, FRG1 is also a developmentally regulated sarcomeric protein suggesting FRG1 may perform a muscle-specific function. Thus, FRG1 is the only FSHD candidate protein linked to the muscle contractile machinery and may address why the musculature and vasculature are specifically susceptible in FSHD. Elsevier B.V. All rights reserved.

Testing the effects of FSHD candidate gene expression in vertebrate muscle development

Mon, 12/22/2014 - 7:30pm

The genetic lesion leading to facioscapulohumeral muscular dystrophy (FSHD) is a dominant deletion at the 4q35 locus. The generally accepted disease model involves an epigenetic dysregulation in the region resulting in the upregulation of one or more proximal genes whose overexpression specifically affects skeletal muscle. However, multiple FSHD candidate genes have been proposed without clear consensus. Using Xenopus laevis as a model for vertebrate development our lab has studied the effects of overexpression of the FSHD candidate gene ortholog, frg1 (FSHD region gene 1), showing that increased levels of frg1 systemically led specifically to an abnormal musculature and increased angiogenesis, the two most prominent clinical features of FSHD. Here we studied the overexpression effects of three other promising FSHD candidate genes, DUX4, DUX4c, and PITX1 using the same model system and methods for direct comparison. Expression of even very low levels of either DUX4 or pitx1 early in development led to massive cellular loss and severely abnormal development. These abnormalities were not muscle specific. In contrast, elevated levels of DUX4c resulted in no detectable adverse affects on muscle and DUX4c levels did not alter the expression of myogenic regulators. This data supports a model for DUX4 and PITX1 in FSHD only as pro-apoptotic factors if their expression in FSHD is confined to cells within the myogenic pathway; neither could account for the vascular pathology prevalent in FSHD. Taken together, increased frg1 expression alone leads to a phenotype that most closely resembles the pathophysiology observed in FSHD patients.

Facioscapulohumeral muscular dystrophy region gene-1 (FRG-1) is an actin-bundling protein associated with muscle-attachment sites

Mon, 12/22/2014 - 7:30pm

In vertebrates, overexpression of facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) recapitulates the pathophysiology exhibited by FSHD patients, although the role of FRG1 in FSHD remains controversial and no precise function for FRG1 has been described in any organism. To gain insight into the function and potential role of FRG1 in FSHD, we analyzed the highly conserved Caenorhabditis elegans ortholog, frg-1. C. elegans body-wall muscles contain two distinct subcellular pools of FRG-1: nuclear FRG-1, concentrated in the nucleoli; and cytoplasmic FRG-1, associated with the Z-disk and costamere-like structures known as dense bodies. Functionally, we demonstrate that FRG-1 is an F-actin-bundling protein, consistent with its localization to dense bodies; this activity is conserved in human FRG1. This is particularly intriguing because it places FRG-1 along side the list of dense-body components whose vertebrate orthologs are involved in the myriad myopathies associated with disrupted costameres and Z-disks. Interestingly, overexpressed FRG-1 preferentially accumulates in the nucleus and, when overexpressed specifically from the frg-1 promoter, disrupts the adult ventral muscle structure and organization. Together, these data further support a role for FRG1 overexpression in FSHD pathophysiology and reveal the previously unsuspected direct involvement of FRG-1 in muscle structure and integrity.

FSHD region gene 1 (FRG1) is crucial for angiogenesis linking FRG1 to facioscapulohumeral muscular dystrophy-associated vasculopathy

Mon, 12/22/2014 - 7:30pm

The genetic lesion that is diagnostic for facioscapulohumeral muscular dystrophy (FSHD) results in an epigenetic misregulation of gene expression, which ultimately leads to the disease pathology. FRG1 (FSHD region gene 1) is a leading candidate for a gene whose misexpression might lead to FSHD. Because FSHD pathology is most prominent in the musculature, most research and therapy efforts focus on muscle cells. Previously, using Xenopus development as a model, we showed that altering frg1 expression levels systemically leads to aberrant muscle development, illustrating the potential for aberrant FRG1 levels to disrupt the musculature. However, 50-75% of FSHD patients also exhibit retinal vasculopathy and FSHD muscles have increased levels of vascular- and endothelial-related FRG1 transcripts, illustrating an underlying vascular component to the disease. To date, no FSHD candidate gene has been proposed to affect the vasculature. Here, we focus on a role for FRG1 expression in the vasculature. We found that endogenous frg1 is expressed in both the developing and adult vasculature in Xenopus. Furthermore, expression of FRG1 was found to be essential for the development of the vasculature, as a knockdown of FRG1 resulted in decreased angiogenesis and reduced expression of the angiogenic regulator DAB2. Conversely, tadpoles subjected to frg1 overexpression displayed the pro-angiogenic phenotypes of increased blood vessel branching and dilation of blood vessels, and developed edemas, suggesting that their circulation was disrupted. Thus, the systemic upregulation of the FRG1 protein shows the potential for acquiring a disrupted vascular phenotype, providing the first link between a FSHD candidate gene and the vascular component of FSHD pathology. Overall, in conjunction with our previous analysis, we show that FRG1 overexpression is capable of disrupting both the musculature and vasculature, recapitulating the two most prominent features of FSHD.

Muscular dystrophy candidate gene FRG1 is critical for muscle development

Mon, 12/22/2014 - 7:30pm

The leading candidate gene responsible for facioscapulohumeral muscular dystrophy (FSHD) is FRG1 (FSHD region gene 1). However, the correlation of altered FRG1 expression levels with disease pathology has remained controversial and the precise function of FRG1 is unknown. Here, we carried out a detailed analysis of the normal expression patterns and effects of FRG1 misexpression during vertebrate embryonic development using Xenopus laevis. We show that frg1 is expressed in and essential for the development of the tadpole musculature. FRG1 morpholino injection disrupted myotome organization and led to inhibited myotome growth, while elevated FRG1 led to abnormal epaxial and hypaxial muscle formation. Thus, maintenance of normal FRG1 levels is critical for proper muscle development, supportive of FSHD disease models whereby misregulation of FRG1 plays a causal role underlying the pathology exhibited in FSHD patients. Developmental Dynamics 238:1502-1512, 2009. (c) 2008 Wiley-Liss, Inc.

Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia

Mon, 12/22/2014 - 7:30pm

The current goal of diabetes therapy is to reduce time-averaged mean levels of glycemia, measured as HbA1c, to prevent diabetic complications. However, HbA1c only explains <25% of the variation in risk of developing complications. Because HbA1c does not correlate with glycemic variability when adjusted for mean blood glucose, we hypothesized that transient spikes of hyperglycemia may be an HbA1c-independent risk factor for diabetic complications. We show that transient hyperglycemia induces long-lasting activating epigenetic changes in the promoter of the nuclear factor kappaB (NF-kappaB) subunit p65 in aortic endothelial cells both in vitro and in nondiabetic mice, which cause increased p65 gene expression. Both the epigenetic changes and the gene expression changes persist for at least 6 d of subsequent normal glycemia, as do NF-kappaB-induced increases in monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 expression. Hyperglycemia-induced epigenetic changes and increased p65 expression are prevented by reducing mitochondrial superoxide production or superoxide-induced alpha-oxoaldehydes. These results highlight the dramatic and long-lasting effects that short-term hyperglycemic spikes can have on vascular cells and suggest that transient spikes of hyperglycemia may be an HbA1c-independent risk factor for diabetic complications.

Engineered telomeres in transgenic Xenopus laevis

Mon, 12/22/2014 - 7:30pm

The expanding roles of telomeres in epigenetic gene regulation, nuclear organization, and human disease have necessitated the establishment of model organisms in which to study telomere function under normal developmental conditions. We present an efficient system for generating numerous vertebrate animals containing engineered telomeres using a Xenopus laevis transgenesis technique. Our results indicate Xenopus zygotes efficiently recognize telomeric repeats at chromosome break points and form telomeric complexes thus generating a new telomere. The resulting transgenic animals progress through normal development and successfully metamorphose into froglets despite the chromosome breakage. Overall, this presents an efficient mechanism for generating engineered telomeres in a vertebrate system and provides an opportunity to investigate epigenetic aspects of telomere function during normal vertebrate development.