The MYC oncogene has been implicated in the regulation of up to thousands of genes involved in many cellular programs including proliferation, growth, differentiation, self-renewal, and apoptosis. MYC is thought to induce cancer through an exaggerated effect on these physiologic programs. Which of these genes are responsible for the ability of MYC to initiate and/or maintain tumorigenesis is not clear. Previously, we have shown that upon brief MYC inactivation, some tumors undergo sustained regression. Here we demonstrate that upon MYC inactivation there are global permanent changes in gene expression detected by microarray analysis. By applying StepMiner analysis, we identified genes whose expression most strongly correlated with the ability of MYC to induce a neoplastic state. Notably, genes were identified that exhibited permanent changes in mRNA expression upon MYC inactivation. Importantly, permanent changes in gene expression could be shown by chromatin immunoprecipitation (ChIP) to be associated with permanent changes in the ability of MYC to bind to the promoter regions. Our list of candidate genes associated with tumor maintenance was further refined by comparing our analysis with other published results to generate a gene signature associated with MYC-induced tumorigenesis in mice. To validate the role of gene signatures associated with MYC in human tumorigenesis, we examined the expression of human homologs in 273 published human lymphoma microarray datasets in Affymetrix U133A format. One large functional group of these genes included the ribosomal structural proteins. In addition, we identified a group of genes involved in a diverse array of cellular functions including: BZW2, H2AFY, SFRS3, NAP1L1, NOLA2, UBE2D2, CCNG1, LIFR, FABP3, and EDG1. Hence, through our analysis of gene expression in murine tumor models and human lymphomas, we have identified a novel gene signature correlated with the ability of MYC to maintain tumorigenesis. The targeted inactivation of oncogenes may be a specific and effective treatment of cancer. However, how oncogene inactivation leads to tumor regression is not clear. Previously, we have shown that even the brief inactivation of the MYC oncogene can result in the sustained regression of at least some tumors. To understand the mechanism, we have utilized several novel genomic analyses to define a set of genes that strongly correlate with the ability of the MYC oncogene to maintain tumorigenesis. First, we generated a novel data set from microarray analyses of murine tumors that we analyzed by StepMiner to identify discrete step changes in gene expression after the inactivation or the reactivation of the MYC oncogene. Second, we utilized Boolean Network Analysis to further define the subset of genes highly correlated with MYC in human tumorigenesis. Third, we utilized ChIP analysis to demonstrate that in many cases the permanent changes of gene expression we uncovered were associated with changes in the ability of MYC to occupy the promoter locus. Our general strategy could be similarly utilized in other experimental model systems to understand how specific oncogenes contribute to the maintenance of tumorigenesis. Overexpression of MYC is one of the most frequent events in human tumorigenesis http://www.myc-cancer-gene.org), it is hard to discern which of the many of MYC targets are associated with the ability of MYC to initiate and/or sustain tumorigenesis.MYC is thought to play a role in the regulation of up to 15% of genes in the fly, mouse or human Many previous studies have examined changes in gene expression associated with the induction of MYC expression in cells in vitroThe MYC induced osteosarcoma derived cell line, 1325, was grown cDNA microarray analysis was performed on the RNA samples prepared from tumors in which MYC was inactivated and reactivated for different lengths of time. StepMiner analysis  [29] wasRecently, we have shown that MYC inactivation generally induces cellular senescence in several tumor models Generally, analysis of gene expression changes after StepMiner analysis revealed four discrete patterns of changes in gene expression upon MYC inactivation and reactivation: Permanently Repressed (PR), Permanently Induced (PI), Reversibly Repressed (RR) and Reversibly Induced (RI). For this analysis, we set p<0.01 as a cutoff for statistically significant changes in gene expression. We identified 1016 unique probes in the PR group, 1777 unique probes in the PI group, 1148 unique probes in the RI group, and 1167 unique probes in the RR group  to the list of genes generated above. Biological functions that were identified for each step upon MYC inactivation are listed , and S5.Notably, MYC has been shown to regulate expression of ribosomal structure proteins and ribosomal RNAs www.myc-cancer-gene.org, a collection of most of the published MYC target genes in different organisms and tissues MYC has been shown to regulate the gene expression of a multitude of genes An important recent report suggests that MYC binding to promoters is regulated by the chromatin structure at these gene loci versus the MYC reactivated conditions (−4). Similarly, the majority of the genes with the PR pattern of gene expression exhibited a significant decrease of MYC binding to E-boxes relative to the MYC ON condition  when MYC was reactivated , and MYC off in pancreatic tumors for 2, 4, and 6 days (referred as tumor regression arrays in the published paper). MYC activation induced pancreatic tumors and MYC inactivation resulted in tumor regression through apoptosis To determine if the gene signature we identified would also be seen in another tumor model system, we compared our microarray data from MYC-induced osteosarcoma with a previously reported microarray data set from a MYC-induced pancreatic tumor model to identify a common expression signature for MYC-induced tumorigenesis We first used the StepMiner algorithm was applied to the raw data generated from these published experiments to obtain lists of genes that increase (or decrease) in expression upon tumorigenesis and decrease (or increase) in expression upon tumor regression . After Set al. RECOMB 2007 in press, see MYC overexpression has been implicated in the pathogenesis of many types of human cancer, in particular, hematopoietic tumors −5, The Boolean analysis identified that the expression of both small and large ribosomal structural proteins is high in human lymphomas  and S4 aFinally, we validated our results obtained by microarray analysis through quantitative real-time PCR . MoreoveMYC target genes have been implicated in a multitude of biological functions To identify this gene signature, we utilized our conditional transgenic model system of MYC-induced osteosarcoma in which we have previously shown that upon MYC inactivation tumors permanently lost the ability of MYC to induce tumorigenesis Drosophila, the biological connection of MYC and ribosomal structural proteins can also be seen in the small cell-size phenotypes of both MYC mutants and ribosomal structural protein genes mutants Our results are consistent with a multitude of evidence suggesting that MYC can regulate ribosomal gene expression Interestingly, changes in the gene expression of ribosomal structural proteins, although observed in both our model of MYC induced osteosarcoma and lymphoma, were not seen in a model of pancreatic islet cell tumors . Thus, iGenes that we identified as most strongly correlated with MYC-induced tumorigenesis  in mice We were able to utilize our MYC conditional tumor models as tools to uncover genes that are strongly correlated with tumor maintenance. However, we recognize that it is very unlikely that any of the individual genes we identified are sufficient alone to explain the ability of MYC to initiate or maintain tumorigenesis. Rather it is highly likely that it is a constellation of gene expression changes that are responsible for the ability of MYC to maintain tumorigenesis.We can now offer a possible explanation for why the brief inactivation of MYC can result in the permanent loss of the ability of MYC to sustain tumorigenesis One of the biggest challenges in understanding how MYC contributes to tumorigenesis has been to address the conundrum that MYC has both direct and indirect influence on the expression of so many different genes and these genes are involved in a multitude of biologic functions. Many of these genes may not be relevant to how MYC overexpression contributes to tumorigenesis. Here we have illustrated by using a defined transgenic mouse model that exhibits conditional tumorigenesis such that upon MYC inactivation tumor cells permanently loses a neoplastic phenotype that we can define a specific gene list that is specifically correlated with MYC's ability to maintain tumorigenesis. To perform this analysis we combined two novel methods of gene expression analysis, the StepMiner and the Boolean analysis, as a powerful strategy to perform an unbiased comparative analysis of microarray data from conditional MYC-induced tumor models and all the available published human data with Affymetrix U133A format. Our strategy may be generally useful for the identification of gene signatures associated with the ability of specific oncogenes to initiate and sustain tumorigenesis and the identification of potential new therapeutic targets for the treatment of cancer.−4% of 2-mercaptoethanol (Sigma). 20ng/ml of doxycycline was added to the medium for inactivating MYC expression. Seven times, each time with 20 mls of PBS, was applied to cells to completely remove doxycycline in the medium.Osteosarcoma-derived cell line 1325 35S35S (PerkinElmer) per plate for 60 minutes and then washed with PBS. Cells were lysed and TCA precipitation was applied to determine the incorporation of radiolabeled amino acids. Aliquots of cell lysate were used for protein determination by DC Protein Assay (Bio-Rad). The protein synthesis rate was calculated as TCA-precipitable counts per minute divided by micrograms of protein in the same sample.For rate of protein synthesis, lymphoma-derived cell line 6780 cDNA were synthesized by Superscript II (invitrogen) followed by manufacture's protocol. Real-time PCR for human c-MYC (probes and primers from Applied Biosystems) and mouse GAPDH Mouse cDNA microarrays were produced at Stanford Functional Genomic Facility. cDNA labeling and hybridization were followed as previously described The StepMiner fits step functions to time-course microarray data and provides a statistical measure of the goodness of fit ChIP was performed based on the protocol provided in the kit with some modifications (ChIP assay kit by Upstate Biotech). Briefly, bone tumor cells were grown on the condition described above with (MYC OFF and MYC reactivated conditions) or without (MYC ON condition) doxycycline (20ng/ml). 48 hours treated with doxycycline, cells were either harvested (as MYC OFF condition) or extensively washed with PBS (see above) to remove doxycycline in the medium. 48 hours after washing, cells were harvested (as MYC reactivated).http://frodo.wi.mit.edu/)  was added to the medium to a final concentration of 1% for cross-linking at 37°C for 10 minutes. Cross-linking was stopped by adding glycine to a final concentration of 0.125M. Cells were washed with cold PBS containing protease inhibitors  and pelleted by centrifugation. Cell pellets were then lysed in SDS lysis buffer . Cells were sonicated with a Branson 250 sonicator at a power setting of 3 for 3 times with 10 sec for each sonication and the cells were cooled down with ice for 1 min between each sonication. This condition of sonication yielded genomic DNA fragments with a size about 100–600 base pairs. Samples were then immunoprecipitated with c-MYC antibody (2 μg of N262 from Santa Cruz Biotech) followed the protocol provided by the kit (Upstate Biotech). DNA samples from the ChIP experiments were applied for quantification by Real-time PCR (ABI PRISM 7900 HT) with SYBR green. Promoter sequences (−2000 to +2000 relative to the transcription start sites) of murine MYC targets were retrieved from UCSC genome browser and primers flanking the E-box were designed by Primer3 (it.edu/) .t + 0.5 are classified as “high,” expression levels below t−0.5 are classified as “low,” and values between t −0.5 and t+0.5 are classified as “intermediate” . Two hundred and seventy three different human Lymphoma microarray experiments were identified using a simple string search “Lymphoma” in the GEO description of the experiment. Genes that are “high” or “low” in more than 95% of the Lymphoma experiments were automatically discovered. Human homologs of genes which were associated with MYC-induced tumorigenesis in mice were selected for this manuscript.Data from 7,171 publicly available raw Affymetrix U133A human microarrays were collected from the Gene Expression Omnibus (GEO) Figure S1Western blot for MYC protein expression upon MYC inactivation and reactivation. Western blots for anti-MYC and α-tubulin antibodies (1.21 MB EPS)Click here for additional data file.Figure S2Induced and repressed patterns of gene expression upon MYC inactivation in osteosarcomas identified by the StepMiner algorithm. MYC OFF arrays  were selected for the StepMiner analysis to retrieve significant changes as the “induced” and “repressed” patterns. Statistically significant changes (p<0.01) after StepMiner analysis are shown here. Genes previously shown to be associated with senescence are highlighted.(1.75 MB EPS)Click here for additional data file.Figure S3High expression of both MYC and small ribosomal structural proteins in human lymphomas by Boolean analysis. MYC expression (Y axis) and small ribosomal structural protein expression (X axis) were plotted for all the published human microarrays in U133A format (green marks). Red lines represent the cutoff thresholds and blue lines mark the “intermediate” regions. Green marks represent all human arrays and red marks represent arrays from lymphoma tissues.(29.83 MB EPS)Click here for additional data file.Figure S4High expression of both MYC and large ribosomal structural proteins in human lymphomas by Boolean analysis. MYC expression (Y axis) and large ribosomal structural protein expression (X axis) were plotted for all the published human microarrays in U133A format (green marks). Red lines represent the cutoff thresholds and blue lines mark the “intermediate” regions. Green marks represent all human arrays and red marks represent arrays from lymphoma tissues.(35.18 MB EPS)Click here for additional data file.Figure S5Validation of mRNA expression of gene signature in MYC-induced osteosarcomas by quantitative real-time PCR. Quantitative real-time PCR with primers specific for genes associated with MYC induced tumorigenesis were applied to cDNA samples upon MYC inactivation for 0, 8, 24, 48 and MYC reactivation for 8, 24 and 48 hours in MYC induced osteosarcomas to validate the mRNA expression. mRNA expression was normalized by r-tTA expression. Relative expression levels were shown here (MYC inactivation for 0 hour was reset to 1).(3.99 MB EPS)Click here for additional data file.Figure S6Validation of mRNA expression of gene signature in MYC-induced lymphomas by quantitative real-time PCR. Quantitative real-time PCR with primers specific for genes associated with MYC induced tumorigenesis were applied to cDNA samples upon MYC inactivation for 0, 6, 12, 24, 48 hours in MYC-induced lymphomas. mRNA expression was normalized by r-tTA expression. Relative expression levels were shown here (MYC inactivation for 0 hour was reset to 1).(3.08 MB EPS)Click here for additional data file.Figure S7Examples of Boolean analysis for expression of BUB1B vs. CCNB2 and XIST vs. RPS4Y1 in the U133A format. Both BUB1B and CCNB2 were found to be expressed in a cell cycle dependent manner (1.86 MB EPS)Click here for additional data file.Table S1List of LUIDs, gene names and Genbank accession numbers of induced and repressed genes upon MYC inactivation.(0.24 MB XLS)Click here for additional data file.Table S2List of LUIDs, gene names and Genbank accession numbers of genes permanently repressed (PR), permanently induced (PI), reversibly repressed (RR), and reversibly induced (RI) upon MYC inactivation.(0.37 MB XLS)Click here for additional data file.Table S3GO term analysis of genes permanently induced or repressed upon MYC inactivation in osteosarcoma. Permanently induced (PI) genes (expression went up and stayed up) and permanently repressed (PR) genes upon MYC inactivation was analyzed by GO Term to identify possible representative biological processes in each time points along the time-course for MYC inactivation. Statistically significant (p<0.01) biological processes in each step are listed here .(0.04 MB DOC)Click here for additional data file.Table S4Gene Ontology (GO) term analysis for genes with PI patterns upon MYC inactivation. Permanently induced (PI) genes (expression went up and stayed up) upon MYC inactivation was analyzed by GO Term to identify possible representative biological processes in each time points along the time-course for MYC inactivation. Statistically significant (p<0.01) biological processes in each step .(0.06 MB XLS)Click here for additional data file.Table S5Gene Ontology (GO) term analysis for genes with PR patterns upon MYC inactivation. Permanently repressed genes (expression went down and stayed down) upon MYC inactivation was analyzed by GO Term to identify possible representative biological processes in each time points along the time-course for MYC inactivation. Statistically significant (p<0.01) biological processes in each step .(0.08 MB XLS)Click here for additional data file.Table S6List of gene names with the PR and PI patterns for three previously published reports regarding direct MYC targets (0.09 MB XLS)Click here for additional data file.Table S7Raw data of ChIP experiments. ChIP for MYC binding to E-box regions were examined for ribosomal structural proteins  and gene(0.21 MB XLS)Click here for additional data file.Table S8List of gene names which were induced (repressed) in tumorigenesis (MYC activation) and repressed (induced) in tumor regression (MYC inactivation) in MYC-induced murine pancreatic tumors. The StepMiner algorithm was applied to the raw microarray data generated from the pancreatic tumors upon MYC activation and MYC inactivation (0.04 MB XLS)Click here for additional data file.Table S9List of Affymetrix probe IDs of human homologs of genes associated with MYC-induced tumorigenesis in mice.(0.02 MB XLS)Click here for additional data file.Table S10List of primer sequences and promoter sequences for quantitative real-time PCR and ChIP.(0.65 MB XLS)Click here for additional data file.Table S11List of numbers of E-box sequences in promoter regions (−2000 to +2000) of common murine MYC target genes. Promoter sequences were retrieved for common murine MYC target genes shown in (0.02 MB XLS)Click here for additional data file.


Although bycatch of industrial-scale fisheries can cause declines in migratory megafauna including seabirds, marine mammals, and sea turtles, the impacts of small-scale fisheries have been largely overlooked. Small-scale fisheries occur in coastal waters worldwide, employing over 99% of the world's 51 million fishers. New telemetry data reveal that migratory megafauna frequent coastal habitats well within the range of small-scale fisheries, potentially producing high bycatch. These fisheries occur primarily in developing nations, and their documentation and management are limited or non-existent, precluding evaluation of their impacts on non-target megafauna.−1, rivaling that of oceanwide industrial-scale fisheries, and threatening the persistence of this critically endangered population. As a result of fisher participation in this study and a bycatch awareness campaign, a consortium of local fishers and other citizens are working to eliminate their bycatch and to establish a national loggerhead refuge.30 North Pacific loggerhead turtles that we satellite-tracked from 1996–2005 ranged oceanwide, but juveniles spent 70% of their time at a high use area coincident with small-scale fisheries in Baja California Sur, Mexico (BCS). We assessed loggerhead bycatch mortality in this area by partnering with local fishers to 1) observe two small-scale fleets that operated closest to the high use area and 2) through shoreline surveys for discarded carcasses. Minimum annual bycatch mortality in just these two fleets at the high use area exceeded 1000 loggerheads yearBecause of the overlap of ubiquitous small-scale fisheries with newly documented high-use areas in coastal waters worldwide, our case study suggests that small-scale fisheries may be among the greatest current threats to non-target megafauna. Future research is urgently needed to quantify small-scale fisheries bycatch worldwide. Localizing coastal high use areas and mitigating bycatch in partnership with small-scale fishers may provide a crucial solution toward ensuring the persistence of vulnerable megafauna. North Pacific loggerheads nest exclusively in Japan, and annual censuses indicate as much as a 90% decrease in nesting females within the past three generations to fewer than 1000 yr−1 skiff−1, particularly while fishing bottom-set gear. Entangled and hooked turtles are generally drowned, and carcasses are discarded at sea Caretta caretta) in the small-scale fisheries of BCS and compared it with their bycatch in industrial-scale pelagic fisheries.Although the impact of small-scale fisheries on this population has not been quantified, reports indicated that juvenile loggerheads aggregate off of Baja California Sur, Mexico (BCS) exposing them to mortality in coastal fisheries operating from small (6–8 m) skiffs up to 55 km offshore hotspots) and compared these with small-scale fishing grounds. We satellite-tracked 30 loggerhead turtles (curved carapace length (CCL); 72±9 cm, mean±SD; 6 km2 across the North Pacific, turtles generally used a relatively small region during the 5,594 turtle days observed , where an average 0.65±1.3 loggerheads were caught per deep fishing trip.We assessed loggerhead bycatch mortality by partnering with local fishers to observe two small-scale fleets that operated closest to the high use area. One fleet fished bottom-set gillnets (Puerto López Mateos) and the other bottom-set longlines . Twenty-four loggerheads were landed dead or died shortly thereafter, resulting in an observed bycatch mortality rate of 92% during longline.In September 2005, we observed 26 loggerheads in seven longline day-trips  . Loggerhminimum annual loggerhead bycatch in each small-scale fishing fleet as the product of the observed mean of turtles killed per boat per day, the minimum number of boats fishing per day, and the minimum number of days fished per year. We estimated that in the 2005 season at least 299 and 680 loggerhead turtles died in the observed bottom-set gillnet and longline fleets, respectively. Our minimum estimate of total loggerhead mortality during 2005 in just two small-scale fishing fleets thus approached 1000 turtles. Although the estimates of minimum annual bycatch for the fleets we sampled are based on a limited number of fishing trips, actual loggerhead mortality for the region is likely to be much higher because 1) we used minimum values for all factors except bycatch rates, for which we used observed point estimates and 2) we estimated annual bycatch for two among twelve or more fleets which fish in or near the loggerhead high use area.We estimated We also conducted daily (May-September) and weekly (October-April) shoreline surveys from 2003–2005 along the 43 km Playa San Lázaro, BCS, which is adjacent to the observed fishing grounds. Nearly 80% (N = 781) of the 982 loggerhead carcasses encountered were observed from May-September, corresponding to seasonal operation of local small-scale fisheries . CarcassThe long-term tracks of loggerhead turtles presented here plus the observed mortality confirm preliminary identification of a high use area for juvenile loggerhead turtles in the coastal waters of Baja California Sur, Mexico minimum annual loggerhead mortality due to bycatch in just two local BCS fleets is more than an order of magnitude greater, we conclude that these two fleets alone may threaten the persistence of the North Pacific loggerhead population.The US National Marine Fisheries Service noted that 37–92 large juvenile North Pacific loggerheads killed per year would “appreciably increase their extinction risk” −1) for the two small-scale fleets rivals that of North Pacific industrial-scale fisheries. For example, the international pelagic driftnet fishery killed an estimated ∼800 loggerheads yr−1 until it was banned by international accord in 1992 −1 across the entire Pacific Basin, roughly half of which (1300 loggerheads yr−1) may be killed in the North Pacific Our minimum bycatch estimate (∼1000 loggerheads yr−1) was also more than an order of magnitude higher than that recorded for industrial-scale fisheries (0.01 turtles gillnet km−1) Bycatch per unit effort (BPUE) was at least an order of magnitude higher in the small-scale longline fleet (19.3 turtles per thousand hooks) than in Mexican and US pelagic longline fleets (0.00–1.40 loggerheads per thousand hooks) Because small-scale fisheries are conducted primarily in developing nations where management and enforcement are limited, assessing and mitigating their bycatch presents an international conservation challenge. Command-and-control approaches such as fisheries closures are often impractical and inadvisable, particularly in developing nations Accordingly, we forged innovative partnerships with local fishers and their families to assess and mitigate their bycatch This case study demonstrates that a co-management strategy that directly engages local fishers and their families holds considerable promise in assessing and mitigating small-scale fisheries bycatch. Mexico is recognized worldwide for its successful protection of gray whales, and it has established numerous marine protected areas along the Baja California peninsula. The establishment of a co-managed loggerhead refuge would greatly reduce the extinction risk of this endangered population.While bycatch in industrial-scale fisheries has driven declines in marine megafauna, small-scale fisheries can apparently have similarly severe effects where they overlap with megafauna high use areas. New telemetry studies are revealing that a range of migratory megafauna spend considerable time in coastal waters during vulnerable life history stages Small-scale gillnet and longline fisheries are ubiquitous to coastal waters worldwide Although mitigating small-scale fisheries bycatch presents a daunting conservation challenge, the high BPUE of these fisheries provides an unexpected advantage. For each unit of small-scale fishing effort modified to reduce bycatch, a much higher benefit accrues for the megafauna than might be expected for industrial-scale fisheries. Localizing coastal distributional hotspots of vulnerable megafauna will be important for identifying previously unquantified bycatch mortality. Protecting coastal hotspots in partnership with local fishers may provide unforeseen leverage for ensuring the persistence of endangered marine megafauna.The movements of loggerhead turtles were monitored using platform transmitting terminals (PTT) deployed on loggerhead turtles (n = 30) released along the Pacific coast of Baja California Sur (BCS), Mexico from 1996–2005. Twenty-seven of these turtles were captured by hand from small fishing boats and released within 18 hours and 10 km of capture. Two turtles were retrieved from bottom-set longlines on which they were shallowly hooked, instrumented, and released as above. One turtle was retrieved from gillnet fishers in the Gulf of California and held in captivity for 10 years before release.−1. Positions of location quality Z and those that clearly fell outside each turtle's track were omitted. In order to preserve the highest spatiotemporal resolution of the data, consecutive ARGOS hits were linearly interpolated to 3 positions per day based on great circle distances, based on the observed mean of 2.7±2.9 hits/day.We attached PTTs to turtle carapaces using polyester resin and fiberglass cloth Multi-individual hotspots off the BCS coast were determined through an effort-weighted kernel density analysis of 9244 filtered positions to derive an index of turtle residence probability per unit area. From our dataset of filtered and interpolated positions, we derived an index of turtle residence probability per unit area as follows: 1) we extrapolated the number of turtle days spent per 0.01°×0.01° cell using kernel density analysis with a search radius of 0.5° and 2) we weighted the kernel density estimate of turtle days spent in each cell by multiplying it by the number of individual turtles using that cell. In this way we downweighted cells frequented by single or few individuals for extended periods to avoid biasing our identification of multi-individual high-use areas. We present turtle residence probability per cell as utilization distributions (UD) based on polygon coverage using least squares cross validation From June to July 2005, we made 73 day-long bottom-set gillnet trips with 5 fishing crews of Puerto López Mateos, the fishing community closest to the loggerhead high use areas identified in this study. Each boat fished a total of fifteen days across the fleets' range of gillnetting depths, with five days in each of three depth ranges that spanned the fleet's fishing grounds  to standardize for boat-specific bycatch rates. Gillnet captains were compensated MN$500 per day, roughly 2/3 of daily gasoline expenditure for bycatch observations to be made.The gillnet crews of Puerto López Mateos reportedly fish daily from May through August (70 to 110 day trips) in depths ranging from 5 to 45 m, with nets soaked for 20–48 hrs. Fishers worked from 6–8 m outboard-powered skiffs and targeted primarily California halibut  and used 20.3 cm mesh monofilament gillnets of 400 m length and height from 3 m to 6 m. All nets were fished with “suspenders” connecting the float line to the sink line resulting in loose bags of net material. The fleet numbered up to 75 boats in 2005, with 9 to 40 boats fishing the fleet's deep area on a given day (32–45 m depths).In September 2005, we made 7 daylong bottom-set longline trips with 5 local fishing crews from Santa Rosa. Longlines targeted shark species and were anchored in 60 to 90 m depths and checked each day. Crews checked and baited an average of 200 hooks per day.The longline crews of Santa Rosa reportedly fished daily from August through September in 2005 (40 to 60 day trips), targeting primarily demersal sharks, with lines soaked 20–48 hours. Fishers used freshly caught mackerel or bycaught tuna or marlin for bait on “Japanese J-hooks” with inflected shanks. The Santa Rosa fleet numbered 5 to 6 boats in 2005.In both fisheries we recorded the number, species, condition, and measurements of sea turtles captured.From January 2003 through December 2005, we conducted shoreline surveys on daily (May-September) and weekly (October-April) schedules along the 44 km Playa San Lázaro the shoreline closest to the loggerhead high use area described here. All turtle carcasses encountered were identified, measured, and marked. Data recorded on each stranded carcass included the following: observer name, stranding date, species, turtle number by day, location, curved carapace length and width (CCL and CCW), condition of carcass (decomposition state), tag numbers (if present), sex of carcass , and observer notes. Curved carapace length was taken from the nuchal notch to the posterior marginal tip. Curved carapace width was taken at the widest part of the shell. All animals were painted and dragged well above the high tide line to avoid recounts.minimum annual bycatch mortality to be 299 loggerhead turtles for the 2005 season of the observed gillnet fleet*. For the bottom-set longline fishery, we multiplied the mean observed number of turtles caught per boat per day (3.7) by the minimum number of boats fishing  by the minimum number of days fished per boat in 2005  and discounted by the proportion of turtles released alive (8%). We thus extrapolated a minimum annual bycatch mortality rate of 680 loggerhead turtles in the 2005 season for the observed bottom-set longline fleet*. Our minimum estimate of the total loggerhead mortality during 2005 for the two small-scale fishing fleets observed totaled ∼1000 loggerhead turtles.For the gillnet fishery, because bycatch was highly dependent on fishing depth, we multiplied the observed mean number of turtles caught per boat per day in depths from 32–45 m (0.65), by the reported minimum number of boats working waters deeper than 32 m on a given day  by the minimum number of days fished per year  and discounted by the proportion of turtles released alive (27%). Based on this simple calculation we extrapolated a The estimates of minimum annual bycatch and observed BPUE for the two fleets we sampled are based on relatively small numbers of fishing trips because of the inherent difficulties of documenting bycatch in small-scale fisheries 3 and 69×103 hooks observed respectively Gillnet bycatch estimates and BPUE for the Japanese pelagic driftnet fleet was published based on 25,500 km of observed gillnet sets Despite our relatively small sample sizes, we are confident that our samples are representative because 1) in semi-structured interviews we conducted in 2003, local fishers reported an average of 4 loggerheads caught per week per boat (roughly 0.65 per day-trip) and 2) in informal interviews made during this study longline fishers reported that the observed bycatch rates were normal. Furthermore, longline observations made both prior to and following this study showed similarly high bycatch rates.Grupo Tortuguero, a community conservation network that unites fishers and other conservationists of the Baja California peninsula and beyond We partnered with fishers, community members, and managers to assess habitat use and bycatch and to design and conduct experiments to reduce turtle bycatch Table S1(0.03 MB DOC)Click here for additional data file.Table S2(0.10 MB DOC)Click here for additional data file.


Autism spectrum disorders represent a group of developmental disorders with strong genetic underpinnings. Several cytogenetic abnormalities or de novo mutations able to cause autism have recently been uncovered. In this study, the literature was reviewed to highlight genotype–phenotype correlations between causal gene mutations or cytogenetic abnormalities and behavioural or morphological phenotypes. Based on this information, a set of practical guidelines is proposed to help clinical geneticists pursue targeted genetic testing for patients with autism whose clinical phenotype is suggestive of a specific genetic or genomic aetiology. Diagnostic and statistical manual of mental disorders, 4th edition (DSM-IV) criteria for pervasive developmental disorders, including autistic disorder, Asperger syndrome, childhood disintegrative disorder, Rett syndrome and “pervasive developmental disorder not otherwise specified” (PDD-NOS).7Autism spectrum disorders (ASDs) represent a heterogeneous group of neurodevelopmental disorders characterised by social and communication deficits, accompanied by repetititive and stereotyped behaviours, with onset before 3 years of age.MeCP2 gene,9Genetics strongly contributes to the pathogenesis underlying ASDs.BRCA1 and BRCA2 mutations,Genetic screens represent a powerful tool when dealing with monogenic mendelian disorders, characterised by direct genotype–phenotype correlations. In the case of complex disorders, such as ASD, widespread genetic testing would not only be expensive and time-consuming, but also generally inappropriate due to the aetiological complexity described above.During the past few years, genetic research in ASDs has begun achieving some important successes, including the identification of several vulnerability loci and of a few cytogenetic abnormalities or single-base mutations able to cause autism (see below). In order to transfer this recently acquired knowledge into clinical practice, it is critical to define a set of phenotypic inclusion criteria that must be met by affected probands to justify their enrolment in a specific genetic testing programme. Accordingly, these genetic screening programmes must be designed to use clearly targeted, feasible and cost-effective strategies. We reviewed the relevant literature to look for specific correlations between ASD-causing gene mutations or cytogenetic abnormalities and clinical ASD phenotypes . We hope this information will be useful to guide clinical geneticists in establishing and implementing effective genetic screening programmes for those patients with ASD whose phenotype is suggestive of a specific genetic or genomic aetiology.Syndromic autism (ASD associated with a known cause) represents approximately 10% of all ASD cases and is often associated with malformations and/or dysmorphic features.16–et alet alet alet al22–The recent advent of array-based high-resolution genomic analysis has dramatically increased the power to detect small de novo genomic deletions and duplications, down to a few kilobases in size, well below the threshold of detection by standard chromosomal banding and karyotyping techniques. At least four independent genome-wide studies have identified de novo microdeletions, microduplications or copy number variants (CNVs) associated with autism using array-comparative genome hybridisation (CGH) techniques using either bacterial artificial chromosome (BAC) or single-nucleotide polymorphism arrays.18NLGN3 and NLGN4 genes, located at human chromosome loci Xq13 and Xq22.33, respectively, have been found to host rare mutations explaining <1% of ASD cases . However, V403M and R704C were also found in unaffected siblings, suggesting they could represent rare non-synonymous variants rather than true mutations. None of 553 patients with ASD assessed in five other studies were found to carry NLG3 or NLG4 non-synonymous variants.34–et al.39Neuroligins are cell-adhesion molecules localised postsynatptically in both glutamatergic  and gamma-aminobutyric acid-ergic (NLGN2) synapses.SD cases . Jamain  et al28–. LaumonnThe clinical phenotype of human NLG mutation carriers is very heterogeneous. In both pedigrees carrying the D396X and R451C mutations, two affected brothers carrying the mutated gene were diagnosed, one with Asperger syndrome (the “speech-preserved” variant of autism) and the other with autistic disorder. The extended pedigree carrying the D429X frameshift mutation in NLGN4 encompasses 10 male carriers affected with non-specific X-linked MR, two male carriers with autism and one with PDD-NOS . Individuals carrying microdeletions involving the NLGN4 may even satisfy diagnostic criteria for neuropsychiatric disorders apparently unrelated to ASDs.In summary, NLG mutation carriers display a variety of syndromes, ranging from X-linked MR without autism, to Asperger syndrome, autistic disorder of variable severity and PDD-NOS. Disease onset may be insidious or abrupt and regressive. These patients do not share any morphological or dysmorphic feature apt to spur a specific diagnostic investigation. The low frequency of these mutations and the lack of phenotypic traits suggesting the involvement of NLG3 and NLG4 do not encourage the clinical implementation of widespread genetic screens for these genes in patients with non-syndromic ASD.SHANK3 gene is located on the telomeric terminal of chromosome 22q13.3 and encodes for a scaffolding protein found in the postsynaptic density (PSD) complex of excitatory synapses, where it binds directly to neuroligins. Two recent studies have shown a possible correlation between mutations or small cytogenetic rearrangements affecting SHANK3 and an ASD phenotype mainly characterised by severe verbal and social deficits (SHANK3 mutation screening. Five of seven cases reported to date carry deletions (142 kb to 4.36 Mb in size), of the terminal 22q13 region encompassing SHANK3, whereas the other two have a frameshift (E409X) and a missense mutation (Q321R).The deficits .40 41 SiSHANK3  have two independent promoters, which determine two mRNA classes: long mRNAs, encoding for α-neurexins and short mRNAs, encoding for β-neurexins.et alNRXN1 gene were also shown to be paternally inherited in one patient.NRXN1 gene was found in two affected sisters, who both received a diagnosis of autism.et al,Presynaptic neurexins induce postsynaptic differentiation in contacting dendrites, by interacting with their postsynaptic neuroligin partners.The presence of deletions or other mutations also in unaffected siblings of patients with autism and even in controls,MECP2 gene located on chromosome Xq28 occur in 80% of female patients with Rett syndrome, a pervasive developmental disorder generally characterised by regression, autism, microcephaly, stereotyped behaviours, epilepsy and breathing problems, whereas in males, mutations are generally lethal.NLGN3 and MECP2, but is not significantly skewed in ASD families overall.47Methyl-CpG-binding protein 2 (MeCP2) is a transcriptional repressor that binds to methylated CpG dinucleotides generally located at gene promoters and recruits HDAC1 and other proteins involved in chromatin repression.MECP2 gene for mutations in patients with non-syndromic ASD , a frameshift mutation (1157del41) and a nonsense mutation (R294X).51–MECP2 mutations are rare in the autistic population, accounting for 0.8–1.3% of cases in the female ASD population  people with macrocephaly and autism . Macrocephaly has been consistently found in approximately 20% of patients with autism recruited in independent samples.71–PTEN de novo mutations in these macrocephalic/macrosomic patients with ASD can be estimated at 4.7% (6/126) (PTEN-related cancers during adulthood.Cases of ASD with  (6/126) . ImportaBased on the information summarised above, we have defined a set of practical guidelines that clinical geneticists should consider, together with recently reviewed ethical issues,If a couple has a non-syndromic autistic child, the probability that a second child will also be autistic is approximately 5–6%.A karyotype and fragile-X testing must be requested for all patients with ASD.Clinicians should be aware that prenatal genetic testing typically raises unrealistic expectations in parents of children with autism because, with the exception of rare gross chromosomal rearrangements, the genetic or genomic anomalies responsible for ASD are not detectable using current prenatal screening methods.In the presence of dysmorphic features and evident neurological symptoms, it is reasonable to suspect chromosomal rearrangements even if the karyotype appears normal. Depending upon availability and cost, a BAC or oligo array-based CGH analysis is strongly advised in these cases. As these technologies will become progressively more available, it will be important not to restrict them to patients with dysmorphia, as microdeletions and microduplications are also common among patients with idiopathic, non-dysmorphic ASD.NLG3 and NLG4 mutations do not share any specific clinical features and the frequency of these mutations is sufficiently low that widespread screening for neuroligin mutations do not seem to be clinically justified.Patients with For the same reasons and owing to the small number of studies performed to date, widespread screening for the neuroxin genes are also not advisable at present.SHANK3 gene can potentially be implemented for patients with severe verbal and social deficits, bearing in mind that some SHANK3 mutations have also been found to be transmitted to unaffected siblings of ASD probands .HOXA1 mutations are recessive. It is thus reasonable to screen only patients from consanguineous families, when presenting with autism associated with deafness, troncoencephalic anomalies and especially horizontal gaze abnormalities.PTEN gene should be screened for mutations in all children with autism and extreme macrocephaly (⩾3 SD). Because of the tumour susceptibility produced by PTEN haploinsufficiency, it is strongly advised to start an oncological monitoring programme immediately for patients with ASD carrying PTEN mutations.The If the parents of a macrosomic autistic child (carrying no genomic rearrangement or mutation affecting the genes discussed above) decide to have another child, clinicians should monitor weight, height and head circumference of the baby sibling at regular intervals from birth to 2 years of age . If the neonate displays a progressive overgrowth of head and body size compared with age-specific and sex-specific population norms, it is advisable to start an early intervention programme, wherever available, with the aim of stimulating social cognition at 18–24 months of life.The design of behavioural intervention programmes targeted to address ASD signs and symptoms as early as possible  is being actively pursued in many clinical centres, in order to intervene during the critical period of maximum plasticity in postnatal brain development.


Imatinib, a tyrosine kinase inhibitor has revolutionized the therapy of Philadelphia chromosome positive chronic myeloid leukemia. Side effects of imatinib include grade 1-4 hepatotoxicity in a subset of patients. We report the case of a 46-year-old male with chronic myeloid leukemia, who developed hepatic hemosiderosis during treatment with imatinib. After ruling out the established congenital and acquired causes of hepatic hemosiderosis, we attribute this to a possible side effect of imatinib therapy. This condition was successfully treated with periodic phlebotomy thus precluding discontinuation of imatinib. To our knowledge, this is the first report of hepatic hemosiderosis most likely consequent to imatinib therapy. Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence of Philadelphia chromosome, which is the translocated 9:22 chromosome giving rise to constitutively active BCR-Abl fusion protein ,2.Imatinib mesylate, an orally administered tyrosine-kinase inhibitor, targets this fusion protein and produces anti-proliferative and pro-apoptotic effects on the neoplastic cells containing the translocation. Reported hepatic side effects observed with imatinib range from mild elevation of transaminases, to acute fulminant hepatitis and fatal liver failure -4,7. We This is the case of a 46-year-old Caucasian male who came to us with complaints of mild fatigue and unexplained weight loss of about 28 pounds over 8 month period. Physical exam was remarkable for splenomegaly without any lymphadenopathy. Labs revealed leucocytosis with a white cell count of 63.3 × 103/μl. A manual differential count of peripheral blood smear showed neutrophil 44%, lymphocyte 1%, monocyte 3%, eosinophil 1%, basophil 6%, bands 22%, metamyelocyte 14%, myelocyte 9%. Following diagnosis of chronic phase CML by bone marrow biopsy the patient was started on 400 mg imatinib daily. Within a month of treatment, his white counts dropped from about 63,000/μl to 12,000/μl. Cytogenetic tests showed typical 9:22 translocation in all 20 banded cells examined. 4 of these cells also had an additional Philadelphia chromosome indicating a possible clonal evolution suggestive of accelerated or blast phase crisis of the disease. Thus the dosage of imatinib was increased to 600 mg daily. In another couple of weeks, his white cell count dropped to 3400/μl alongwith a drop of hemoglobin from 13.4 gm/dL to 11.1 gm/dL and platelets from 239,000/ μl to 57,000/ μl. Further drop of WBC count to 1800/ μl over the next 10 days warranted a reduction of imatinib dosage from 600 mg to 400 mg daily. His white count however continued to drop to 1500/ μl when he became febrile and was advised to discontinue imatinib for a couple weeks. His fever resolved with antibiotics and following 2 weeks of discontinuation of imatinib, his white count was back up at 5100/ μl, with absolute neutrophil count of 2700/ μl, hemoglobin 13.9 and platelets 190,000/ μl. The patient was restarted on 400 mg of imatinib daily. He tolerated this dose well for 2 weeks without any drop in cell counts and the imatinib dosage was increased to 500 mg daily. This dose was continued thereafter.By the first 9 months following initial diagnosis the patient was able to achieve and maintain a complete molecular remission as evident by quantitative RT-PCR (reverse transcriptase polymerase chain reaction) of BCR-Abl transcript. His splenomegaly had resolved clinically. It was concerning however that his liver function tests became increasingly abnormal. Although his baseline liver function tests were within normal limits prior to treatment initiation, his ALT  was noted to be mildly elevated one month after the start of imatinib therapy, became normal after nine months and then again progressively increased to markedly high levels at eighteen months . On the The discovery of the BCR-Abl translocation in CML culminated in the realization of the dream of targeted therapy in cancer by the small molecule tyrosine kinase inhibitor imatinib . The recIn the absence of transfusional iron overload, hepatic hemosiderosis in the aforesaid case with normal transferrin saturation is a possible consequence of imatinib therapy. The elevated liver enzymes were not associated with any discernible infectious or inflammatory condition hence making hemophagocytosis a less likely possibility. While anti-nuclear antibody (ANA) was elevated at 2.3 OD ratio (ref range <1.5 OD ratio), anti-mitochondrial antibody, anti-smooth muscle antibody and alfa-1-antitrypsin antibody were negative. It seems unlikely that the mechanism of iron overload is a result of increased iron absorption or simply a higher intake in way of iron being an ingredient in imatinib mesylate tablets since there was no evidence of iron overload in any other organs including heart, spleen or pancreas. Whether it was secondary to interference of imatinib with heme metabolism in the reticuloendothelial cells remains to be elucidated. In this case report, we made an effort to understand the pathology behind imatinib induced hepatotoxicity and were able to treat the condition successfully by periodic phlebotomy. This is relatively safer than using measures as corticosteroid therapy or stopping imatinib treatment as have been shown to be effective in reversing other forms of imatinib induced hepatotoxicity. Imatinib induced disturbance in the iron homeostasis is also suggested by a recent report of intradermal hemosiderosis as a possible consequence to imatinib therapy, lending further support to our conclusion that the observed hepatic hemosiderosis is most likely a consequence imatinib therapy .


Ancestry analysis on chromosome 9q supported an inverse association between the rs2378383 minor allele (G) and childhood asthma. This work identifies chromosome 9q21.31 as a novel susceptibility locus for childhood asthma in Mexicans. Further, analysis of genome-wide expression data in 51 human tissues from the Novartis Research Foundation showed that median GWAS significance levels for SNPs in genes expressed in the lung differed most significantly from genes not expressed in the lung when compared to 50 other tissues, supporting the biological plausibility of our overall GWAS findings and the multigenic etiology of childhood asthma.Many candidate genes have been studied for asthma, but replication has varied. Novel candidate genes have been identified for various complex diseases using genome-wide association studies (GWASs). We conducted a GWAS in 492 Mexican children with asthma, predominantly atopic by skin prick test, and their parents using the Illumina HumanHap 550 K BeadChip to identify novel genetic variation for childhood asthma. The 520,767 autosomal single nucleotide polymorphisms (SNPs) passing quality control were tested for association with childhood asthma using log-linear regression with a log-additive risk model. Eleven of the most significantly associated GWAS SNPs were tested for replication in an independent study of 177 Mexican case–parent trios with childhood-onset asthma and atopy using log-linear analysis. The chromosome 9q21.31 SNP rs2378383 (p = 7.10×10 TLE4 on chromosome 9q21.31 (a novel candidate region for childhood asthma) and replicated one polymorphism in an independent study of childhood-onset asthmatics with atopy and their parents of Mexican ethnicity. Hispanics have differing proportions of Native American, European, and African ancestries, and we found less Native American ancestry than expected at chromosome 9q21.31. This suggests that chromosome 9q21.31 may underlie ethnic differences in childhood asthma and that future replication would be most effective in populations with Native American ancestry. Analysis of publicly available genome-wide expression data revealed that association signals in genes expressed in the lung differed most significantly from genes not expressed in the lung when compared to 50 other tissues, supporting the biological plausibility of the overall GWAS findings and the multigenic etiology of asthma.Asthma is a leading chronic childhood disease with a presumed strong genetic component, but no genes have been definitely shown to influence asthma development. Few genetic studies of asthma have included Hispanic populations. Here, we conducted a genome-wide association study of asthma in 492 Mexican children with asthma, predominantly atopic by skin prick test, and their parents to identify novel genetic variation for childhood asthma. We implicated several polymorphisms in or near  Asthma (OMIM 600807) is a leading chronic childhood disease with prevalence rates reaching a historically high level (8.9%) in the United States and continuing to increase in many countries worldwide ORM1-like (S. cerevisiae)  and gasdermin B  with risk of childhood asthma in German and British populations ORMDL3 and GSDML SNPs with risk for childhood asthma across various populations phosphodiesterase 4D, cAMP-specific   with risk of asthma in whites from the United States and replicated this finding in two other white populations Genome-wide association studies (GWASs) have emerged as a powerful approach for identifying novel candidate genes for common, complex diseases. In the first asthma GWAS, using 307,328 single nucleotide polymorphisms (SNPs), Moffatt et al. found highly statistically significant associations of SNPs in adjacent genes Few genetic studies of asthma have included Hispanic populations, and replication of positive genetic findings is scarce across Hispanic groups. Hispanics have differing proportions of Native American, European, and African ancestries. This admixture introduces special considerations (such as controlling for population stratification in association studies), but admixture in Hispanic populations also provides a unique opportunity to use ancestry analysis to evaluate our genetic association findings Mexico City is one of the most polluted cities in the world, and its inhabitants experience chronic ozone exposure, which has been linked to asthma development in children and adults and asthma exacerbations In the GWAS, there were more male (58.7%) than female (41.3%) children with asthma, and the mean age at enrollment was 9.0±2.4 years . The repAdditional demographic and clinical characteristics of the 492 children with asthma evaluated in the GWAS are presented in The 520,767 autosomal SNPs passing quality control were tested for association with childhood asthma using additive modeling with the log-linear method in 492 children with asthma and their biological parents from Mexico City. Not surprisingly given the study size, no SNP met genome-wide significance using a conservative Bonferroni adjustment. Nonetheless, the comparison of observed and expected p-values in the quantile-quantile plot  shows se−6)], followed by an intronic SNP in potassium voltage-gated channel, Shab-related subfamily, member 2  on chromosome 8 [rs2247572 (p = 1.94×10−6)] and two intergenic SNPs on chromosome 20 [rs6063725 (p = 3.52×10−6)] and rs720810 (p = 5.13×10−6)] with only moderate linkage disequilibrium (LD) (r2 = 0.59). The next most significant SNP (rs2378383) highlights a cluster of SNPs on chromosome 9q21.31 ranking among the top GWAS SNPs. This cluster of SNPs spans transducin-like enhancer of split 4 (E(sp1) homolog, Drosophila)  and its upstream region. LD analysis of SNPs with p≤0.001 in this cluster shows two large LD blocks in this region with one block encompassing TLE4 and the other block encompassing the upstream region (m region .−5 to 1.55×10−6 (Eleven of the 18 most significantly associated SNPs met our criteria to be selected for replication in 177 case-parent trios of Mexican ethnicity from the Genetics of Asthma in Latino Americans (GALA) study .55×10−6 . There w.55×10−6 . The repTLE4 in an intergenic region between coiled-coil-helix-coiled-coil-helix domain containing 9 (CHCHD9) and TLE4, was associated with childhood asthma in the replication study with p = 0.03. Meta-analysis of rs2378383 in the two studies gave a combined p-value of 6.79×10−7, and the RR estimate for carrying one copy of the rs2378383 minor allele (G) compared to carrying no copies in the GWAS  was quite similar to the RR estimate in the replication study . The SNP rs2378377, a neighboring intergenic SNP in moderate LD with rs2378383 (r2 = 0.73), had a marginal association with childhood asthma in the replication study with p = 0.06 (combined p = 2.68×10−6). RR estimates for the rs2378377 minor allele (G) were also similar between the GWAS  and the replication study . None of the other nine SNPs were associated in the replication study  were obtained from the previous GWASs of asthma TLE4 and its upstream region with small p-values, and the SNPs listed above are located in close proximity to many of our associated SNPs. Similar to our GWAS and replication study, only cases with childhood-onset asthma were included in both GWASs in white populations Association results from additive modeling for SNPs in the region spanning Associations of rs2378383 and rs2378377 were examined in data from the GWAS population stratified by residential ambient ozone exposure  and by current parental smoking . The minor alleles of both SNPs were inversely associated with asthma at p<0.05 in all strata (results not shown), thus giving no evidence for effect modification in the presence of these environmental exposures.−7) and rs2378377 (p = 1.08×10−6) being the two top ranking SNPs. In addition to smaller p-values, the magnitudes of association with asthma were slightly stronger for rs2378383  and rs2378377  in the subset of trios where the asthmatic child is also known to be atopic.Among the 445 cases with skin test data available, 408 can be classified as atopic by virtue of having at least one positive skin test. We repeated the GWA scan in this subset of 408 trios. Chromosome 9q21.31 SNPs predominated among the top ranked SNPs, with rs2378383 (p = 7.18×10The chromosome 9q21.31 SNPs rs2378383 and rs2378377 were tested for association with the number of positive skin tests as a quantitative measure of the degree of atopy in the trios with skin test data. Both SNPs were associated with degree of atopy (p = 0.0018 for rs2378383 and p = 0.0010 for rs2378377). Their RR estimates indicate an inverse association in which carrying one copy of the minor allele is associated with a decreasing number of positive skin tests , consistent with the direction of association for asthma.The Mexican subjects in the GWAS had mean ancestral proportions of 69.5±15.6% for Native American, 27.3±14.3% for European, and 3.2±3.0% for African ancestries. Given the predominance of Native American ancestry, we evaluated Native American transmission in the GWAS along the chromosomal arm (9q) containing the replicated SNP (rs2378383) by ancestry analysis. As shown in The deficiency in Native American ancestry at this locus suggests that a protective allele occurred at a higher frequency in the Native American ancestral population than in the European and African ancestral populations. An examination of this SNP in the HapMap and Human Genome Diversity Panel (HGDP) data reveals that the frequency of the G allele is generally low in European, African, and East Asian populations , while its frequency is much higher in Native American populations (0.57 in HGDP Pima and 0.36 in HGDP Mayan). This pattern suggests that the G allele may be tagging a protective allele in the Native American ancestral population. This conclusion is consistent with the finding that the G allele is associated with a decreased risk for childhood asthma in the GWAS and replication analyses .We examined gene expression patterns in 51 diverse human tissues in the context of GWAS findings to determine whether genes expressed in asthma relevant tissues ranked higher than genes not expressed in such tissues and thus to assess the biological plausibility of our overall GWAS findings. These results are presented in TLE4 did not contribute to the observation of significantly lower GWAS q-values in lung-expressed genes, as TLE4 was classified as not expressed. TLE4 displays a nearly ubiquitous expression pattern with similar low intensity levels across many tissues, so even though it is present in the lung, its expression level in the lung did not exceed our 75th percentile threshold to be classified as expressed.Among the 51 tissues, the most significant deviation between the median q-values was found between 3,618 genes expressed in the lung versus 10,712 genes not expressed in the lung  and fetal lung, trachea, tonsil, smooth muscle, and bronchial epithelium at the p<0.05 level (uncorrected). Thus, SNPs in genes more highly expressed in tissues related to the pathogenesis of asthma and allergies tend to give more significant GWAS results than genes more highly expressed in other tissues. These results give biological credibility to our overall GWAS findings and are consistent with the multigenic etiology of asthma.A two-dimensional cluster analysis was conducted to identify the implicated tissues with correlated gene expression patterns. As shown in TLE4 and its upstream region, and two of these SNPs (rs2378383 and rs2378377) were tested for replication in an independent study of 177 case-parent trios of Mexican ethnicity. Despite the small sample size for replication, both SNPs gave p-values close to 0.05 and the same direction and magnitude of association as the GWAS. Neither rs2378383 nor rs2378377 have a known impact on TLE4 expression, but given their location upstream of TLE4, it is possible that these SNPs reside in a TLE4 regulatory region.Genetic studies of asthma are few in Hispanic populations, and to our knowledge, this work presents the first asthma GWAS in Mexicans and the most extensive coverage of genetic variation for an asthma GWAS in any Hispanic population. The GWAS included 492 Mexican case-parent trios. Given the moderate GWAS sample size, no SNP met genome-wide significance. However, the ranking of GWAS SNPs highlighted a potentially important candidate region for childhood asthma susceptibility, chromosome 9q21.31. Several chromosome 9q21.31 SNPs with small GWAS p-values were located in a priori expect that ancestry analysis results would corroborate log-linear association results.Ancestry analysis in this chromosomal region provided supportive evidence that rs2378383 (G) is associated with a decreased risk of childhood asthma in Mexicans. Ancestry and transmission-based association analyses provide complementary but not completely independent lines of evidence. At each SNP, the log-linear method only used parents who were heterozygous in genotype, while ancestry analysis used all parents who are heterozygous in ancestry, including parents who are homozygous in genotype. We did not Our ancestry analysis uses the same principles that underlie admixture mapping and relies on the key assumption of different risk allele frequencies between the ancestral populations, primarily Native American and European in this study. Under this assumption, individuals with disease in the admixed population would be expected to share an excess of ancestry from the population with the highest risk allele frequency at the disease locus Ancestry analysis implicated chromosome 9q21.31 as a chromosomal region that may underlie ethnic differences in childhood asthma. Complex diseases with differing disease prevalence rates in the ancestral populations are most suitable for ancestry analysis The evidence for locus-specific ancestry around rs2378383 has implications for replication. Because rs2378383 (G) occurs at relatively low frequency in European, African and East Asian populations, genetic association studies in these populations are likely to suffer from lack of power at this locus. In contrast, the G allele occurs at moderate frequency in the Native American populations surveyed in HGDP TLE4 and its upstream region. The TLE family of proteins in humans is homologous to the Drosophila Groucho protein, which participates in cell fate determination for neurogenesis and segmentation. The highly conserved structure among the Drosophila Groucho and human TLE gene products suggest similar functions as transcriptional regulators in cell fate determination and differentiation TLE1, TLE2, TLE3, TLE4, TLE5, TLE6), as deposited in the NCBI database. The distinct expression patterns among the TLE genes suggest a complex mechanism in humans involving non-redundant roles for the TLE genes TLE4 gene, in particular, shows ubiquitous expression across many tissues The chromosome 9q21.31 SNPs associated with childhood asthma in the GWAS map to TLE4 are associated with asthma as well as degree of atopy in our data, and their associations with asthma became more pronounced when considering only the asthmatic children with atopy and their parents. These findings suggest that the influence of TLE4 on asthma may be related to its influence on immune system development.TLE interacts with Runt-related transcription factor 3  in a manner that may be directly relevant to asthma. In mice, loss of RUNX3 function results in an allergic asthma phenotype due to accelerated dendritic cell maturation and resulting increased efficacy to stimulate T cells Childhood asthma is a complex disease, and there are likely many susceptibility genes influencing immune system development and asthma in the Mexican population. The examination of GWAS in the context of genome-wide expression illustrated the biological plausibility of our GWAS findings and showed consistency with the involvement of multiple genes. Genes expressed in the lung show association signals that differ most significantly from the association signals from genes not expressed in the lung when compared to 50 other human tissues. The lung represents a major pathogenic site for asthma, and this finding implies that multiple genes expressed in the lung are collectively associated with an increased risk of childhood asthma. Some of the other implicated tissues may represent false positives, but several of the highlighted tissues are biologically plausible for childhood asthma, including trachea, bronchial epithelium, smooth muscle, and immune tissues such as thymus, tonsil, and lymph node.ORMDL3 region ORMDL3. The PDE4D SNPs implicated by Himes et al. Other GWASs have implicated different susceptibility loci. Several SNPs implicated in the first asthma GWAS by Moffatt et al. in the The major limitation of this study is the sample size for the GWAS and replication study. The Mexican population is largely under-studied given its size, and only moderate sample sizes are currently available for the study of asthma genetics. In our study, no SNPs met genome-wide significance, and no replication SNPs met the significance threshold when using a conservative Bonferroni correction for multiple testing. Despite this limitation, top GWAS findings, replication in an independent population, and ancestry analysis taken together implicate a novel region for association with asthma in Mexican children.This study has several strengths. The case-parent trio design and the log-linear analysis protects against bias due to population stratification TLE4, but further work is needed to decipher whether TLE4 or a nearby gene explain the signals from the chromosome 9q21.31 region. Further, childhood asthma is a complex disease with a proposed multigenic etiology, but most single studies will not have sufficient power to examine such complex relationships. Identification of important interacting risk factors in childhood asthma and other complex diseases will require very large sample sizes. This work identifies chromosome 9q21.31 (including TLE4) as a novel candidate susceptibility locus for childhood asthma, suggests that this region may underlie ethnic differences in childhood asthma, and emphasizes the presence of multiple genetic risk factors in the complex mechanism leading to childhood asthma.The GWAS and replication association results and the supporting ancestry analysis implicate chromosome 9q21.31 as a novel susceptibility locus for childhood asthma in the Mexican population. This region contains a biologically plausible novel susceptibility gene for childhood asthma, The study protocol was approved by the Institutional Review Boards of the Mexican National Institute of Public Health, Hospital Infantil de Mexico Frederico Gomez, and the US National Institute of Environmental Health Sciences (NIEHS). Parents gave written informed consent for the children's participation, and children gave their assent.Children with asthma (aged 5–17) and their biological parents were recruited between June 1998 and November 2003 from a pediatric allergy specialty clinic at a large public hospital in central Mexico City, Hospital Infantil de Mexico Frederico Gomez. The case-parent trio design protects against bias due to population stratification in this admixed population Children were diagnosed with asthma by a pediatric allergist at the referral clinic based on clinical symptoms and response to treatment 1) was calculated using spirometric prediction equations from a childhood population in Mexico City The clinical evaluation also included skin prick testing to measure atopy and pulmonary function testing, as previously described Measurements of ambient ozone were obtained from the Mexican government's air monitoring station closest to each child's residence (within 5 km). The annual average of the daily maximum 8 hour averages of the ozone level was collected for the year prior to study entry and dichotomized at the median for stratified analyses. Further details on the ozone measurement protocol have been described elsewhere Peripheral blood lymphocytes were isolated from whole blood, and DNA was extracted using Gentra Puregene kits . A total of 498 complete case-parent trios with previously confirmed parentage and sufficient amounts of DNA were genotyped for 561,466 SNPs using the Illumina HumanHap 550 K BeadChip, version 3  at the University of Washington, Department of Genome Sciences. Genotypes were determined using the Illumina BeadStudio Genotyping Module, following the recommended conditions. Results for three unrelated study subjects fell below the genotyping call rate threshold of 95% resulting in exclusion of three trios. The remaining study subjects were genotyped with an average call rate of 99.7%.http://pngu.mgh.harvard.edu/~purcell/plink/) −10 (N = 557), Mendelian errors in more than two families , and heterozygous genotype calls for chromosome X SNPs in more than one male (N = 380). All SNP exclusions were made sequentially in the above order.Quality control analyses were conducted using PLINK  in the final analysis data set.http://code.google.com/p/glu-genetics/) (N = 921) Final SNP-level quality control made exclusions due to one or more discordant genotypes across 14 HapMap replicate samples identified using the Genotyping Library and Utilities application (Subjects of Mexican ethnicity from the GALA study were used for replication testing. The GALA study protocol has been described elsewhere The children with asthma in the GWAS (less than 18 years old) were enrolled at a pediatric allergy clinic, so nearly all had allergic asthma. To maximize comparability with the GWAS, the replication study included only the 177 complete case-parent trios comprising subjects of Mexican ethnicity having childhood-onset (age of onset<18 years) asthma and atopy  and their parents.2<0.9) with other higher-ranked SNPs and MAF>10% were selected for replication. Statistical power to detect associations in the replication study of 177 case-parent trios was calculated for each selected SNP using QUANTO (http://hydra.usc.edu/gxe) SNPs were ranked by GWAS p-value. The top SNP and 10 other top ranking GWAS SNPs were tested for replication in the GALA study. The highest ranking SNP along with 10 other top ranking SNPs with no strong LD  PRISM 7500 Real-Time PCR System using primers and probes from ABI's Assay-by-Demand. The assay was performed under universal conditions, with each reaction containing 3.75 ng DNA, 0.125 µL 40X Assay Mix and 2.5 µL TaqMan Universal PCR master mix brought to a final volume of 5 µL with sterile water. Thermal cycling conditions began at 95°C for 10 minutes and then proceeded with 60 cycles of 92°C for 15 seconds and 60°C for 2 minutes. After the PCR reaction, plates were scanned by the ABI PRISM 7500 PCR system to determine genotypes by allelic discrimination.The log-linear likelihood approach was used to examine associations of individual SNPs with childhood asthma in the GWAS and replication study Robustness to population stratification is a well-known property of the case-parent trio design and transmission-based methods. The log-linear method achieves robustness to population stratification by stratifying on the six possible parental mating types, which are defined by the number of copies of the allele carried by each of the two parents http://pngu.mgh.harvard.edu/~purcell/plink/) http://www.broad.mit.edu/mpg/haploview/) The log-linear method was implemented using the LEM computer program Interesting SNPs from the GWAS and replication study were tested for effect modification with environmental exposures relating to air pollution and environmental tobacco smoking. Data from the GWAS population were stratified by current parental smoking (yes/no) and residential ambient ozone exposure , and the log-linear method was used to test for genetic associations in each stratum.Additional analyses were conducted in the GWAS population using the skin prick testing data. The genome-wide association scan was repeated in the subset of trios with children classified as atopic (one or more positive skin tests) in an effort to reduce phenotypic heterogeneity and thus reduce genetic heterogeneity. Then, an extension of the log-linear method for quantitative traits The program FRAPPE was used to estimate individual ancestry proportions, assuming three ancestral populations: Native American, European, and African a priori, a multiple testing correction was not applied. A trio-based ancestry analysis test was implemented similar to that described in Clarke and Whittemore The program SABER was used to infer locus-specific ancestry in each individual http://symatlas.gnf.org) of genome-wide expression profiles of the protein-encoding transcriptome in many diverse human and mouse tissues and cell lines The Genomics Institute of the Novartis Research Foundation maintains a freely accessible database  software was used to perform this analysis using the continuous expression values in the 15,047 genes with expression data.Figure S1TLE4.LD structure of SNPs in or near (0.08 MB DOC)Click here for additional data file.Figure S2Two-dimensional cluster analysis of genome-wide expression data.(3.72 MB DOC)Click here for additional data file.Table S1Q-values for the 3,618 genes classified as expressed in lung tissue.(2.99 MB DOC)Click here for additional data file.


FMR1, and is well conserved from human to Drosophila. In order to generate a genetic tool to study FMR1 function during vertebrate development, we generated two mutant alleles of the fmr1 gene in zebrafish. Both alleles produce no detectable Fmr protein, and produce viable and fertile progeny with lack of obvious phenotypic features. This is in sharp contrast to published results based on morpholino mediated knock-down of fmr1, reporting defects in craniofacial development and neuronal branching in embryos. These phenotypes we specifically addressed in our knock-out animals, revealing no significant deviations from wild-type animals, suggesting that the published morpholino based fmr1 phenotypes are potential experimental artifacts. Therefore, their relation to fmr1 biology is questionable and morpholino induced fmr1 phenotypes should be avoided in screens for potential drugs suitable for the treatment of FXS. Importantly, a true genetic zebrafish model is now available which can be used to study FXS and to derive potential drugs for FXS treatment.Fragile X syndrome (FXS) is one of the most common known causes of inherited mental retardation. The gene mutated in FXS is named  FMR1 gene Fragile X syndrome (FXS) is one of the most common known causes of inherited mental retardation with a frequency of 1∶4000 males and 1∶6000 females FMRP is a ubiquitously expressed RNA-binding protein, including two KH domains and an RGG box, with high expression levels in brain and testis Fmr1 knock-out mice recapitulate the typical characteristics of FXS, including behavioural abnormalities, learning deficits and audiogenic seizures. Microscopic analysis of brain material from both FXS patients and Fmr1 knockout mice has shown dendritic spine abnormalities Fmr1 knock-out mice Fmr1 knock-out mice using mGluR5 antagonists Fmr1 knock-out hippocampal neurons using two different mGluR5 antagonists The development of mouse models of FXS has facilitated cellular studies on the underlying molecular basis of this loss-of-function disorder et al. reported the use of zebrafish embryos to model FXS fmr1 specific MOs resulted in abnormal axonal branching, changes in trigeminal ganglion number and craniofacial abnormalities. Most of these abnormalities in zebrafish embryos could be rescued using MPEP, an mGluR5 antagonist, or by fmr1 overexpression In 2006, Tucker fmr1 knock-out alleles using TILLING . TILLING combines random induced mutations by ENU treatment and subsequent screening for null mutations fmr1 knock-down study In the present study, we generated two independent fmr1 (ENSDARG00000037433), leading to the introduction of a premature stop at codon position 113 . Furthermore, using a C-terminal antibody we are unable to detect expression of Fmr in neurons using immunocytochemistry on paraffin sections, whereas a high expression could be detected in neurons from wild type zebrafish. In order to develop a genetic model in which the effects of FMRP on brain development can be easily studied during development we screened for knock-out alleles in the zebrafish system. From a randomly mutagenized library we isolated two independent mutant alleles: hu2787 defines a C to T change in the coding region of tion 113 . The mRNAnimals lacking zygotic Fmr are found at Mendelian frequencies in crosses between heterozygous parents. They display wild-type development, and develop into fertile adults with no gross abnormalities. Progeny from homozygous mutant parents were also analyzed to check the potential effect of maternally provided protein and/or mRNA on development. Also these maternal-zygotic (MZ) mutant animals develop normally, and display no obvious defects in behaviour or fertility. Importantly, we did not observe selective pressure against homozygous mutant combinations in any of the crosses we performed (not shown), strongly suggesting that potentially lethal phenotypes are not repressed by the presence of genetic modifiers in our genetic backgrounds.fmr1 knock-down studies that have been published before axial, dlx-2a and islet-1. We therefore analyzed the expression of these genes by in situ hybridisation in fmr1 MZ null embryos. The results of these experiments are depicted in fmr1 null embryos in any of the analyses.The results described above contrasts with morpholino induced In addition, we measured the width of Meckel's cartilage and the angle it makes to the anterior-posterior axis, to address whether these mutant animals develop abnormalities that may be related to the craniofacial defects seen in fragile X patients, as described in the morpholino knockdown morphants et alfmr1 morphant embryo's. We therefore analyzed the branching frequency of the Rohon-Beard neurons, similar to what was reported by Tucker et alfmr1 stop mutant animals.Finally, Tucker fmr1 knockout alleles in zebrafish, and as such provide a new genetic model system to study FXS, a highly prevalent form of inherited mental retardation. FXS is caused by the loss of the gene product of fmr1, Fmr. FXS models have been described in multiple systems and from these models it has become clear that FMRP is acting at the synapse to regulate the translation of target mRNAs upon group 1 mGluR stimulation and whose protein products mediate synaptic strength Fmr1 knock-out mice exhibit exaggerated translation of target mRNAs at the synapse. Potentially, such a process can be well affected by mGluR antagonists that would ameliorate the phenotypic outcome of FXS We describe the generation of two fmr1 (fmr1 morphant) in the zebrafish led to embryonic phenotypes that could in principle be used as a read-out in drug screens et al.fmr1 knock down fmr1 function in neurite branching and number of trigeminal ganglion neurons. Furthermore, it suggested that small molecule drug screens in the zebrafish may indeed be an affective manner of finding bio-active lead-compounds that are good starting points for developing drugs beneficial for FXS patients.Establishing a zebrafish model for FXS is very useful in this context, as the zebrafish embryo is amenable to large scale, small molecule drug screens. Supporting this idea was the finding that morpholino induced knock-down of fmr1 knock-down, and this again could be rescued by treatment with MPEP. This is a curious finding, since the role of synaptic connections between neurons in cranial cartilage development is to date totally unexplored, and hence it is not clear whether indeed MPEP would be expected to affect craniofacial defects caused by loss of Fmr.Tucker et al fmr1 mutant alleles in the zebrafish, both of which lead to loss of detectable Fmr. In contrast to the above-mentioned MO study, however, we cannot find any gross phenotypical effects caused by these alleles. We checked specifically the above-mentioned phenotypes, craniofacial abnormalities and neurite branching phenotypes, but find no significant differences between wild type and mutant siblings. Fmr1 mutant fish are also completely fertile, and incrosses between homozygous mutant males and females result in normally developing embryos, indicating that maternally provided mRNA and/or protein is not rescuing first generation fmr1 mutants.We here characterize two fmr1 null animals do not display these defects? First we explore why genetic mutation of fmr1 may miss FXS-relevant phenotypes. Redundancy could potentially be an issue. The morpholinos used could affect the fmr1 homologues fxr1 and fxr2, which are both present in zebrafish. This seems unlikely, however, given the fact the sequence comparison between the morpholino and fxr-1/2 genes shows very little complementarity. Fmr1 itself could be duplicated in zebrafish. However, the most recent genome annotation shows no indication of a duplicated fmr1 gene, and on western blot we detect no protein in fmr1 homozygous mutant tissue. This makes the presence of a closely related, functional fmr1 copy unlikely. Finally, potential phenotypes may be rescued by modifier loci; loci that genetically interact with fmr1 and of which particular alleles may suppress phenotypes triggered by loss of Fmr. Despite the fact that our zebrafish strains show no sign of selection for or against homozygous fmr1 mutants, this is an option that is difficult to eliminate. Extensive outcrossing into the zebrafish strains used in the studies by Tucker et alWhat could be causing the observed phenotypes in the morphants dlx-2a, fgfr1 and axial expression could only be observed in 17/30, 11/30 and 3/30 fmr1 morphants, respectively; for neurite branching phenotypes no numbers are given related to the penetrance of the defect; injection of antibodies against alpha-acetylated tubulin resulted in a dramatic axon defect only in 3/30 and axon defasciculation in 13/30 fmr1 morphants. Finally, the craniofacial dysmorphology could only be observed in 9/15 fmr1 morphants.There is, however, a more likely potential explanation: the morpholino-induced phenotypes may not be related to loss of Fmr. Morpholino oligonucleotides are well known to cause phenotypes unrelated to knock-down of the intended gene. In fact, 15–20% of MOs used in zebrafish show off-targeting effects that are mediated by p53-induced apoptosis fmr1 in zebrafish at most induces very subtle phenotypes that are not readily detectable using light-microscopy and techniques like immunocytochemistry and in situ hybridisation, at least in the strains used in our laboratory. It remains well possible that subtle defects are induced by lesions in fmr1, and that these may be used to develop sensitive and robust essays to probe fmr1 function, which may in turn be used for screening of small molecules libraries in order to find drugs suitable for treatment of FXS. At present, however, we have to conclude that the phenotypes as described by Tucker et alfmr1 function in the zebrafish.In summary, we find the loss of et al. Adult zebrafish were bred and maintained under standard conditions. Staging of embryos was according to Kimmel fmr1 gene. Amplicons were designed for exon 5–6 and exon 7–9 and screened for mutation as described fmr1hu2787 (stop); hu2898fmr1 (splice)) were outcrossed against TL and crossed to obtain homozygous or transheterozygous embryos.ENU induced mutation library was screened for a mutation in the Adult zebrafish were sacrificed by euthanasia using high dose of MS222, brains were dissected immediately and fixed overnight in 3% paraformaldehyde. The brains were embedded in paraffin according to standard protocols. Sections (7 µm) were deparaffinized, followed by antigen retrieval using microwave treatment in 0.01 M sodium citrate solution. Endogenous peroxidase activity blocking and immunoincubation was performed as described before using polyclonal rabbit 758 antibodies against zebrafish Fmrp 2, 100 µM CaCl2, 0.45% Triton X-100 and 0.05% Tween-20, pH 7.6, with Complete protease inhibitor cocktail (Roche Diagnostics), while kept on ice. After incubating the homogenates on ice for 30 minutes, they were sonicated twice for 20 seconds. Cell debris was spun down and the supernatant was collected. Loading mix was added to 100 µg of protein, heated at 95°C for 5 minutes and loaded onto a 10% SDS-PAGE gel. After electroblotting the gel onto a nitrocellulose membrane, the membrane was incubated overnight at 4°C with the rabbit polyclonal 758 antibody specific for zebrafish FMRP Half brains  from adult zebrafish were homogenised in 500 µl HEPES-buffer  from which the cDNA fragment was cloned into pCS2plus;dlx-2a hu2787 fmr1 allele was done in one batch, so that wild-type, heterozygous and homozygous mutant embryos received identical treatments. Embryos were genotyped afterwards, revealing a consistent loss of fmr1 mRNA in homozygous hu2787 mutants.ISH experiment were performed as described in Thisse et al. fmr1hu2787 incross embryos (5 dpf) were Alcian blue stained according to Neuhauss et al. For whole-mount immunohistochemistry, embryos were fixed in 4% paraformaldehyde for four hours at RT, washed with PBT and incubated overnight at 65°C in FST solution . Next day, washed in PBT and blocked in ABS  and incubated overnight with the monoclonal antibody zn-12 . Primary antibody was washed off by ABS buffer, and embryos were incubated overnight with secondary antibody goat anti-mouse conjugated with Alexa-488 . Embryos were imaged with a Leica DM6000 microscope, Leica camera DFC 360 FX and Leica LAS AF Software. Images were analysed using the NeuronJ plugin


Codon optimization of DNA vectors can enhance protein expression both by enhancing translational efficiency, and by altering RNA stability and export. Although gag mRNA in live T cells, we transfected Jurkat cells with increasing doses of capped, polyadenylated synthetic mRNA corresponding to wildtype or codon-optimized gag sequences, measured Gag production by quantitative ELISA and flow cytometry, and estimated the translational efficiency of each transcript as pg of Gag antigen produced per µg of input mRNA. We found that codon optimization yielded a small increase in gag translational efficiency (approximately 1.6 fold). In contrast when cells were transfected with DNA vectors requiring nuclear transcription and processing of gag mRNA, codon optimization resulted in a very large enhancement of Gag production.To quantify translational efficiency of gag translational efficiency per se, with the vast majority of enhancement in protein expression from DNA vectors due to altered processing and export of nuclear RNA.We conclude that suboptimal codon usage by HIV-1 results in only a slight loss of  The lack of nuclear stability and export in the absence of Rev is partly due to the presence of defined inhibitory sequences  within the structural genes themselves. In addition, the low GC content of the HIV RNA also contributes to nuclear instability, even in the absence of defined inhibitory sequences HIV gene expression is controlled by multiple complex regulatory mechanisms gag DNA, Gag expression is greatly enhanced by codon optimization gag RNA, and it is not known to what degree codon usage per se affects translational efficiency of HIV transcripts.For HIV-uninfected cells that are transfected with gag sequences, and transfected Jurkat cells by electroporation in the presence of increasing amounts of synthetic mRNA. This method introduces mRNA directly to the cytoplasm, thus bypassing the requirement for mRNA transcription, processing and nuclear export. We have previously shown that over 95% of cells subjected to electroporation under similar conditions express the mRNA-encoded product To compare the translational efficiency of different transcripts in the cytoplasm of transfected cells for the present study, we generated synthetic mRNA encoding codon-optimized or non-optimized gag. Capped, polyadenylated mRNA was transfected into Jurkat cells by electroporation. Gag protein production was quantified by measuring the concentration of Gag p24 in cell supernatant by ELISA and by staining transfected cells with Gag-p24 specific fluorescent antibody. We found that the amount of Gag released into the culture medium was proportional to the amount of mRNA added to the transfection reaction (2>0.99). The amount of Gag detected by ELISA was proportional to the median fluorescence intensity (MFI) of cells stained with p24-specific fluorescent antibody (2 = 0.93), demonstrating that the amount of secreted Gag detected in cell supernatants was indicative of protein production by the majority of cells in the population.To measure Gag production by transfected cells, we generated synthetic mRNA encoding HIV-1 reaction 2>0.99. antibody 2 = 0.93gag sequences derived from an HIV-1 Clade C clinical isolategag sequences under control of the CMV promoter. For optimized vector, we used the previously described plasmid p96ZM651gag-optTo compare the translational efficiency of different transcripts, we used codon optimized or non-optimized in vitro transcription to generate capped, polyadenylated synthetic mRNA. Jurkat cells were transfected with increasing amounts of mRNA, and the amount of Gag in the supernatant after 24 hours was measured by ELISA. The experiment was repeated for four separate batches of synthetic mRNA, and the relative translational efficiency for each transcript was estimated by calculating the slope of the curve (pg p24/ µg mRNA). To measure the actual translational efficiency of optimized and nonoptimized mRNA delivered directly to the cytoplasm, we constructed transcriptional template vectors encoding identical open reading frames as p96ZM651gag-opt or p96ZM651gag-NonOpt2 above, and performed 7G(5′)ppp(5′)G cap analog. Translational efficiency of synthetic mRNA can be enhanced by the use of an anti-reverse cap analog (ARCA) 7G(5′)ppp(5′)G cap analogs. To validate our comparison of translational efficiencies we compared the effect of codon optimization to the effect of another method of enhancing translational efficiency. For experiments described above transfections were carried out using mRNA capped with conventional mgag translational efficiency. Our results are consistent with the conclusions of previous reports that for DNA-transfected cells, the majority of enhancement in protein expression by gag codon optimization is in fact due to increased stability and export of nuclear mRNA. Nevertheless, we find a reproducible enhancement (in four out of four experiments) of Gag production of approximately 60% that can be attributed to codon optimization. We propose that this value represents the actual difference in translational efficiency due to suboptimal codon usage by clinical-isolate HIV-1, although we also cannot rule out a possible contribution of altered cytoplasmic mRNA stability based on available data. Why a virus that has evolved to replicate in human cells would maintain suboptimal codon usage is unexplained. It is worth noting that the cellular editing enzyme APOBEC3G has a cytidine deaminase activity that can induce G»A hypermutation in the sequence of replicating HIV Constructs bearing codon-optimized genes are widely used to enhance heterologous expression of lentiviral gene products. However, the term “codon optmization” is potentially confusing, in that it implies that enhancement of expression is directly related to codon usage and to translational efficiency. Here we report the first quantitative assessment of the effect of codon optimization on HIV-1 Finally we note that the combined effects of codon optimizaton and ARCA cap shown in  yielded gag sequences were derived from plasmids p96ZM651.8 (GenBank Accession #AF286224), which contains the near full length molecular clone of an HIV-1 subtype C clinical isolate from Zambia; and p96ZM651gag-opt (GenBank accession #AY181195), which contains a codon-optimized gag sequence of p96ZM651 cloned into EcoRI and BamHI cloning sites, 5′→3′ of vector pcDNA3.1(-). p96ZM651gag-opt and p96ZM651.8 were provided by the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH  with identical 5′ and 3′ untranslated regions to p96ZM651gag-opt.The non-optimized and optimized in vitro transcription (IVT) of gag mRNA were constructed by cloning the codon-optimized and non-optmized gag sequence into template vectors consisting of: T7 polymerase promoter, Kozak ribososme binding site, gag open reading frame, 64 nt poly-adenosine tail, AscI restriction site for linearization. All plasmids were sequenced before use to confirm correct sequences.Templates for 6 cells were washed twice with OptiMem (Gibco) and resuspended in 300 µl of OptiMem with the indicated concentration of mRNA or plasmid DNA, left on ice for five minutes, and transfected by electroporation with a GenePulserII (Biorad) at 900V/0.75mSec in a 4 mm cuvette. Cells were then cultured in RPMI plus 10% fetal calf serum for 24 hours, after which conditioned medium was harvested for analysis by p24 ELISA kit (PerkinElmer).Synthetic mRNA was transcribed from the template plasmids using Message Machine T7 or (T7 Ultra kit for ARCA-capped mRNA) (Ambion). mRNA was purified with RNEasy kit (Qiagen) and mRNA concentration was quantified with serial dilutions of mRNA solutions using a ND-1000 spectrophotometer (Nanodrop). For each transfection, 10Transfected cells were cultured in RPMI plus 10% fetal calf serum for 24 hours (RNA transfection) or 48 hours (DNA transfection), after which conditioned medium was harvested for analysis by p24 ELISA kit (PerkinElmer). Cell pellets were fixed and permeablized using Fix/Perm kit , stained with anti-p24 antibody (KC-57-PE Beckman Coulter), and analyzed on a BD FacsCalibur flow cytometer. MFI was determined by gating on all live cells.