Few studies assessing the relationship between active and passive smoking and tuberculosis have used biomarkers to measure smoke exposure. We sought to determine the association between active and passive smoking and LTBI in a representative sample of US adults and children.We used the 1999–2000 US National Health and Nutrition Examination Survey (NHANES) dataset with tuberculin skin test (TST) data to assess the association between cotinine-confirmed smoke exposure and latent tuberculosis infection (LTBI) among adults ages ≥20 years (n = 3598) and children 3–19 years (n = 2943) and estimate the prevalence of smoke exposure among those with LTBI. Weighted multivariate logistic regression was used to measure the associations between active and passive smoking and LTBI.LTBI prevalence in 1999–2000 among cotinine-confirmed active, passive, and non-smoking adults and children was 6.0%, 5.2%, 3.3% and 0.3%, 1.0%, 1.5%, respectively. This corresponds to approximately 3,556,000 active and 3,379,000 passive smoking adults with LTBI in the US civilian non-institutionalized population in 1999–2000. Controlling for age, gender, socioeconomic status, race, birthplace (US vs. foreign-born), household size, and having ever lived with someone with TB, adult active smokers were significantly more likely to have LTBI than non-smoking adults (AOR = 2.31 95% CI 1.17–4.55). Adult passive smokers also had a greater odds of LTBI compared with non-smokers, but this association did not achieve statistical significance (AOR = 2.00 95% CI 0.87–4.60). Neither active or passive smoking was associated with LTBI among children. Among only the foreign-born adults, both active (AOR = 2.56 (95% CI 1.20–5.45) and passive smoking (AOR = 2.27 95% CI 1.09–4.72) were significantly associated with LTBI.Active adult smokers and both foreign-born active and passive smokers in the United States are at elevated risk for LTBI. Targeted smoking prevention and cessation programs should be included in comprehensive national and international TB control efforts. Mycobacterium tuberculosisTuberculosis (TB) remains a substantial global health problem, with an estimated one out of every three people worldwide infected with While active or former smoking has been associated with increased risk for LTBI Passive smoke exposure may contribute to the household spread of TB, and contacts of TB patients who smoke have been shown to have an independent and increased risk of LTBI In many previous studies on the relationship between passive smoking and LTBI researchers have relied on self-reported smoking status to assess risk We sought to determine the association between active and passive smoking and LTBI in a representative sample of the US population using cotinine verification of smoking exposure among non-smoking adults and children.The National Health and Nutrition Examination Survey(NHANES) is administered in 2 year cycles by the Centers for Disease Control and Prevention's (CDC) National Center for Health Statistics (NCHS) and is a nationally representative sample of the US civilian  population ≥1 year of age, with oversampling of persons aged ≥60 years and minority racial/ethnic groups The NHANES study protocol was approved by the NCHS Institutional Review Board (IRB). Since the current analysis used only de-identified data, it was exempt from IRB review.After TST placement, induration was measured 48–72 hours later by trained readers. Induration ≥10 mm was considered a positive result for LTBI. For those with evidence of Bacillus Calmette-Guérin(BCG) vaccination , induration of ≥15 mm was considered positive. There were 39 adults and 4 children that were BCG-vaccinated and had induration between 10–15 mm that were not considered to have LTBI.Self-reported current smoking behavior was measured through the home interview followed by the lab interview conducted at the time of the physical examination, when serum sampling is completed. At the home interview adults that had smoked at least 100 cigarettes in their life were asked “Do you currently smoke cigarettes?”. At the lab interview adults and adolescents 12–19 years were asked “During the past 5 days, did you use any product containing nicotine including cigarettes, pipes, cigars, chewing tobacco, snuff, nicotine patches, nicotine gum, or any other product containing nicotine?” and if adolescents indicated they had ever tried smoking they were also asked “During the past 30 days, on how many days did you smoke cigarettes?”.Participants were classified as non-, passive, or active smokers using information from both the home and lab interviews and the laboratory results using CDC definitions Covariates of interest included age at time of examination (approximately 4–6 weeks after the home interview), gender, race/ethnicity, country of birth , poverty income ratio , level of education, the number of persons living in the same household, and whether the participant had ever lived with someone with active TB. Frequency of alcohol use among adult participants with complete TST tests and smoking status had a substantial amount of missing data (15.3%); we performed a sensitivity analysis to compare the inclusion of heavy alcohol use (≥5 drinks per day) in the multivariate model for adults.Adults ≥20 years of age and children ≥3-19 were analyzed separately due to potential biological differences in respiratory and immune systems. Additionally, adults and children (aged 13–19) received different questionnaires ascertaining smoking behavior. Weighted analyses were used to account for oversampling of the elderly, and minority race/ethnicity subpopulations. To account for non-response for those without a complete TST reading, the two-year Taylor series weights provided by the NCHS for NHANES were adjusted according to Bennett et al. (see online supplement) Respondents with LTBI were more likely to have been born outside the United States, and have lower household income than those who did not have LTBI . Among aAdults with cotinine-confirmed active smoking status were almost two times as likely to have LTBI  compared to non-smokers . After aThe strongest correlate of LTBI for both adults and children in this sample was foreign-born status .ThereforThe prevalence of LTBI overall was 4.8% (95% CI 3.4%–6.1%) for adults and 1.1% (95% CI 0.3%–1.9%) for children aged 3–19 . The preAfter weighting all data and using serum cotinine to confirm smoking status, 28.2% (95% CI 24.0%–32.5%) of adults and 21.3% (95% CI 16.8%–25.8%) among those 12–19 years of age were active smokers in the United States in 1999–2000; 36.5% (95% CI 33.9%–39.1%) of adults and 58.5% (95% CI 54.5%–62.4%) of children aged 3–19 were passive smokers; and 35.2% (95% CI 30.1%–40.3%) of adults and 31.1% (95% CI 25.6%–36.6%) of children aged 3–19 were non-smokers.Mycobacterium tuberculosis circulating in the community, which is the case for many nations from which the foreign born population in the United States emigrate. In fact, den Boon et al. found that passive smoking was only associated with LTBI among those living with an active TB patient and was not associated among those not living with an active TB patient M. tuberculosis may be an important determinant of the US TB burden.This study of the non-institutionalized US population confirms the positive association between cotinine-confirmed active smoking and LTBI among adult participants. In addition, after stratification by foreign-born status, the risk of LTBI due to active smoking was even greater among adults born outside of the United States and there was no association between active smoking and LTBI among US-born adults. Passive smoking had a marginally significant association with LTBI among US-born adults and a significant association with LTBI among the foreign-born adults. The effect of smoking and smoke exposure may be particularly important where there is a higher prevalence of We did not find that passive smoking was associated with increased risk for LTBI among children in contrast with other published studies on LTBI M. Tuberculosis from the lungs of infected smokers, which could lead to increased transmission.There is increasing mechanistic evidence providing biological plausibility that smoking increases the likelihood of TB infection Since smoking has been associated with poor adherence to treatment for both active TB and LTBI M. tuberculosis infection could not be determined. However, cohort studies needed to identify onset of smoking, onset of LTBI, and length and intensity of exposure as antecedents to LTBI are impractical given the large sample size and long follow-up period necessary. Furthermore, a limitation of using serum cotinine to assess the association between smoking and LTBI is that its half-life is 18–20 hours We used a cross-sectional design for this study; thus, the temporal relationship between smoking and the timing of We used a conservative cutoff point of 10 ng/mL to distinguish active smokers from non-smokers; however, some researchers have recommended even lower cutoff points, ranging from 1–6 ng/mL depending on age and race A challenge of investigating the association between smoking and LTBI in the US population is the low prevalence of LTBI. This may have affected our LTBI risk estimates among children, and, as a result, the confidence limits around our risk estimates for LTBI were quite wide. While the prevalence estimates are more than a decade old, measuring the association between active and passive smoking and LTBI at the US population level may become increasing challenging as the prevalence of smoking, passive smoke exposure and LTBI has decreased since 1999–2000. In addition, the TST test is unable to identify active TB cases from LTBI possibly resulting in an overestimation of LTBI prevalence, though active TB is rare in the United States. Further discussion of the limitations of the TST test and confounders of LTBI in the NHANES study can be found elsewhere While the effects of active and passive smoking on LTBI in the United States across age and country of birth are not consistent in the 1999–2000 NHANES, active smoking among all adults, and active and passive smoking among the foreign-born adults, were strongly associated with LTBI. Further research using cotinine-confirmed smoking exposure assessment, especially for passive smoking, in areas of higher TB prevalence would help to more accurately determine the specific TB burden due to tobacco use. Comprehensive global tobacco control including active and passive smoking prevention and cessation efforts will likely further decrease the prevalence of LTBI in the United States, especially among adults. Tobacco cessation programs among TB patients as well as encouraging smoke-free homes for TB patients have already been recommended by The Union Against Tuberculosis and Lung Disease


This article discusses how extant animal and plant genomes are the result of inherently different rates and modes of genome evolution resulting in relatively stable animal and much more dynamic and plastic plant genomes.Continuing advances in genome sequencing technologies and computational methods for comparative genomics currently allow inferring the evolutionary history of entire plant and animal genomes. Based on the comparison of the plant and animal genome paleohistory, major differences are unveiled in 1) evolutionary mechanisms (i.e Genomes thus represent the foundation from which many fundamental biological insights may be gained. Careful analysis of the genomic content and structure for founder and extinct ancestral karyotypes will further our understanding of the different genomic properties and how they came about in modern species. In particular, the deluge of genomic data, which has dramatically increased in recent years, now offers the opportunity to investigate, for the first time, and in a single analysis using the same methodological approach, paleo-evolutionary patterns and processes that have shaped present-day plant and animal genome organization.Genomes are the blueprints of all living organisms and underpin the mystery of life. Indeed, when considering the presence and absence of genes, expansions, or contractions of gene families, and topologies of Similarities and differences between plant and animal genome structure and evolution have long been a source of intense investigations, mainly based on few genomes comparisons or lacking a unified and transversal approach to perform comparative genomics in both kingdoms . In ordee value, score, and percentage identity. However, the detection of conserved regions is limited when sequence alignments are obtained with these BLAST default parameters. To increase the significance of interspecific sequence alignments for inferring evolutionary relationships between genomes, we used parameters defined from BLAST results  and >50 Mya , respectively  and within a single genome  into contiguous ancestral regions (CAR). Chromosomal blocks that are duplicated in two different genomes but located at orthologous positions when comparing the two genomes are considered as 1) unique in the ancestor , and 2) deriving from a shared prespeciation duplication event. On the contrary, a chromosomal block that is duplicated in one genome but not identified as duplicated at orthologous positions when comparing two genomes is considered as 1) a species-specific duplication, and 2) deriving from a postspeciation duplication event. The same approach is applied for any type of rearrangements including inversions and translocations. From the identified CARs, the most likely evolutionary scenario is proposed on the following assumptions: 1) ancestor modeling is based on duplications (or any shuffling events) found at orthologous positions between modern species, and thus considered as ancestral, 2) evolutionary history is based on the smallest number of shuffling operations  that explain evolution from the ancestral genome to modern karyotypes. Based on these assumptions, the most likely ancestral karyotypes and associated evolutionary scenarios have been proposed for plants and animals.Different approaches such as 1) cladistic , 2) GRIMFinally, the comparison of gene/TE evolutionary dynamics in plants and animals has been investigated based on the genome annotation features published previously  genome sequences are available either as chromosome anchored sequences, unmapped or partial whole-genome sequences, and if not sequenced, at least associated with high-resolution gene-based genetic maps, all allowing evolutionary comparative genomics studies at unprecedented resolution  of ∼25 Mb, structured in five protochromosomes in monocots  went through a WGD , followed by four chromosome fissions (hereafter Cfis) and two fusions (hereafter Cfus) that resulted in an n = 12 ancestral intermediate  Brachypodium evolved through 7 Cfis and 14 Cfus that resulted in a basic number of n = 5 chromosomes. Furthermore, we proposed that the maize genome underwent a recent specific WGD  event, resulting in an intermediate with n = 20 chromosomes, followed rapidly by at least 17 Cfus and 7 Cfis leading to the modern genome structure with 10 chromosomes.The refined characterization of seven paleoduplications and the inference of the relationships between different conserved regions allowed re-evaluating the evolutionary events that have shaped the monocot genomes since their divergence from a putative ancestor with five chromosomes. About 50–90 Mya, the rmediate , right. proposed . We sugg., seven triplicated blocks, all triplicates dating back to ∼200 Mya) in all the eudicot genomes analyzed favors the model with a hexaploid ancestor , with an n = 21 intermediate  common to all eudicots. In this scenario, the grape, fragaria, and cacao genomes all evolved from this intermediate through respectively 2Cfis–4Cfus, 3Cfis–17Cfus, and 2Cfis–13Cfus, to reach their modern genome structure. The poplar and apple genomes subsequently underwent specific WGD  events. The poplar genome structure derived from a Malpighiales intermediate of 12 chromosomes (12 = 21+6Cfis+15Cfus) that have been duplicated (n = 24) with then 4Cfis–9Cfus to reach its n = 19 modern genome structure. The apple genome structure derived from a Rosaceae intermediate of nine chromosomes (9 = 21+3Cfis+15Cfus) that has been duplicated with then 4Cfis–5Cfus to reach its n = 17 modern genome structure. Finally, we propose that the Arabidopsis and soybean genomes evolved from an n = 21 intermediate through two specific WGD . The Arabidopsis thaliana genome structure was derived from a Brassicales intermediate of nine chromosomes (9 = 21+10Cfis+22Cfus) that has been duplicated (n = 18) with then 13Cfus to reach its n = 5 modern genome structure. Finally, we suggest that the soybean genome structure derived from a Papilionoideae intermediate of six chromosomes (6 = 21+1Cfis+16Cfus) that have been duplicated (n = 24) with then 13Cfis–17Cfus to reach its n = 20 extant genome structure, cf. +2Cfus.Similarly, the identification of remnants of an hexaploidy event  that diverged from a common ancestor >450 Mya allowed us to characterize 38,773 orthologous genes defining 737 collinear blocks and covering on average 74% of the considered genomes, showing that between 40% and 50% (for >50 Mya of divergence) and 60–80% (for <20 Mya of divergence) of the genes are conserved as strict orthologs between vertebrate genomes  genomes (available from  genomes .n = 31 Osteichthyes ancestor described below), containing ∼13,000–20,000 protogenes covering a physical gene space size of ∼50 Mb, then complementing alternative inferences of the animal paleohistory  defining 260 paralogous blocks and covering on average 22% of any of the eight animal genomes considered . Integraohistory , as well , followed by three interchromosomal translocations and fusions that resulted in an n = 23 ancestral intermediate (10+10+3 = 23 chromosomes). The n = 23 ancestor went through another WGD . The modern animal genomes derived from a vertebrate common ancestor (Osteichthyes) of 31 protochromosomes resulting from 10Cfus and 1Cfis of the n = 40 Gnathostome intermediate. We propose that a teleost ancestor with 13 protochromosomes derived from the n = 31 vertebrate ancestor intermediate with 23Cfus and 5Cfis. The medaka genome may then have been derived from this n = 13 teleost ancestor including a specific WGD  and additional Cfus (7) and Cfis (5) events. Further, we propose that the chicken, opossum, horse, dog, chimp, human, and mouse genomes have been derived from an Amniote ancestor with 26–30 chromosomes derived from the n = 31 vertebrate ancestor intermediate followed by at least one Cfus. The chicken and opossum genomes derived directly from the n = 30 Amniote clade ancestor with respectively, 1Cfis–3Cfus and 43Cfis–64Cfus to reach their modern n = 33 chromosome and n = 9 chromosome present-day genome structures, respectively. Horse and dog derived from a Laurasiathera intermediate that supposedly had 65 protochromosomes obtained from the n = 30 Amniote clade ancestor plus 57Cfis–22Cfus. The modern horse and dog karyotypes were likely derived from the n = 65 Laurasiathera clade ancestor with respectively, 2Cfis–35Cfus and 6Cfis–32Cfus to reach their modern n = 32 chromosome and n = 39 chromosome present-day genome structures, respectively. Finally, human, chimp, and mouse derived from a common Euarchontoglires intermediate of 39 protochromosomes that have been shaped from the n = 30 Amniote clade ancestor with 24Cfis–15Cfus. The mouse genome structure corresponds to the one of the n = 39 Euarchontoglires intermediate including additional specific Cfus (50) and Cfis (31) events. Chimp and human differ only by a unique chromosome fusion event from their common Hominidae ancestor of 24 chromosomes that has been derived from the n = 39 Euarchontoglires intermediate ancestor with 1Cfis / (17 for chimp and 18 for human) Cfus. About 450 Mya, the Gnathostome ancestor of n = 40 chromosomes. We can speculate that additional WGDs have not been possible in animals once sexual chromosomes have been genetically determined, except for instance in invertebrates, fishes, and amphibians  can now be considered in the context of published studies that discussed consequent impact sexual chromosomes on the reported WDG pattern. Overall, dating of the shared ancestral or specific recent duplication events identifies two distinct types of WGD . We can suggest that the distinct rates of gene order conservation between plants and animals is the consequence of their different evolutionary patterns , angiosperms experienced numerous WGDs from their common paleotetraploid of 12 protochromosomes (resulting from n = 5 of 7 ancestors) or paleohexaploid of 21 protochromosomes (resulting from a n = 7 ancestor) founder genomes, for the monocots and eudicots, respectively. This different rate of gene conservation may be directly linked to massive duplicated gene loss following WGD , genetic bottlenecks via natural selection, metabolic rates, and demography, cannot be excluded to explain such observed difference in genome content conservation between plants and animals  , based on the same analysis framework for both plant and animal genomes, allowed us to investigate the overall genome features that are similar and different in both lineages, at both the genic (current paragraph) and repeat (next paragraphs) levels. We show that within 20 Myr of separation, 10%–20% and 40%–50% of protein-coding genes have been conserved in plants and animals, respectively tables A, right.patterns . While twing WGD  so that wing WGD . Figure ebrates) .Fig. 2.., >35% of Arabidopsis genes in families) compared with animals   illustrates the gene distribution observed for Brachypodium chromosome 1 and human chromosome 16, where gene density increases on plant chromosomes from pericentromeric regions to subtelomeric regions, in human, genes appear in large gene islands (blue peaks), which are absent in plants.We then estimate that, due to their distinct evolutionary histories, modern animal genomes are twice as conserved as plants regarding their gene content compared with plants. Our previous data and associated conclusion regarding differential rate of gene retention showing more evolutionary stability for animal than plant genomes, can directly explain previously published studies that established consequent differences between plant and animal genomes in 1) gene families structure, 2) recombination rate, 3) splicing variants, 4) conserved noncoding sequences, and 5) miRNA retention. The differential pattern of WGD reported here in both lineages has lead to the reported larger multigene families in plant (i.eamilies) . Such deamilies) . The repamilies) . Overallamilies) . Moreoveamilies) , we propamilies)  drives gamilies) . In termamilies) . The repamilies) ; table 2amilies) . At the amilies) . Despiteamilies) . We can B illustrates the TE distribution  observed for Brachypodium chromosome 1 and human chromosome 16. Where TE density is homogenous for human , in plants the nested mode of insertion and reduction by illegitimate recombination produced hot spots of TEs (up to 58% of 500 Kb windows covered by class I TE) separated by low density TE regions . Although class II elements are widespread and active in a variety of eukaryotes, they have been thought to be transpositionally inactive with, no, or few signs of recent activity in mammalian genomes, that is, 3% class II in human compared with plant, 5% class II in Brachypodium.Similar to genes, also transposable elements (TE) can be investigated based on the previously proposed paleogenomics data between the two major eukaryotic lineages to study their differential contribution to genome invasion and contraction. Transposable elements are ubiquitous in eukaryotes  and are typically divided into two classes . Class IFigure 2Alu is the most abundant repetitive element in the human genome) subfamilies continue to be mobile in mammals today (Brachypodium up to 50% in cacao). Overall, ancient, as well as recent Class I LTR TE invasion with a nested mode of insertion so that ancient elements are removed by younger ones through LTR-based illegitimate recombination. Such classical pattern of recent invasion of plant genome may have led to large genome size variation at the interspecific or more interestingly intraspecific levels. Panaud and collaborators reported a 2-fold genome size variation between rice genotypes by the burst of three LTR–retrotransposon families consisting in the accumulation of more than 90,000 retrotransposon copies during the last 3 Myr (copia and gypsy) and non-LTR (LINEs and SINEs) retrotransposons, respectively predominant in plant and animals.The large majority (>55%) of the TEs recognizable in the human genome were inserted prior to the radiation of mammals, ∼80–100 Mya, with an exceptional burst of SINES 40 Mya to reach the present number of more than 1 million of mainly inactive copies . Recent ls today , 2007. Wst 3 Myr , resultist 3 Myr . OverallIn conclusion, comparative analyses reveal the mechanisms that give rise to the different structure and evolution of plant and animal genomes. Polyploidy, more frequent in plants than animals, may trigger genetic and epigenetic changes  of the g


Pufferfishes of the Family Tetraodontidae are the most speciose group in the Order Tetraodontiformes and mainly inhabit coastal waters along continents. Although no members of other tetraodontiform families have fully discarded their marine lives, approximately 30 tetraodontid species spend their entire lives in freshwaters in disjunct tropical regions of South America, Central Africa, and Southeast Asia. To investigate the interrelationships of tetraodontid pufferfishes and thereby elucidate the evolutionary origins of their freshwater habitats, we performed phylogenetic analysis based on whole mitochondrial genome sequences from 50 tetraodontid species and closely related species (including 31 newly determined sequences). The resulting phylogenies reveal that the family is composed of four major lineages and that freshwater species from the different continents are independently nested in two of the four lineages. A monophyletic origin of the use of freshwater habitats was statistically rejected, and ancestral habitat reconstruction on the resulting tree demonstrates that tetraodontids independently entered freshwater habitats in different continents at least three times. Relaxed molecular-clock Bayesian divergence time estimation suggests that the timing of these invasions differs between continents, occurring at 0–10 million years ago (MA) in South America, 17–38 MA in Central Africa, and 48–78 MA in Southeast Asia. These timings are congruent with geological events that could facilitate adaptation to freshwater habitats in each continent. Aquatic environments supply aquatic organisms with a large variety of habitats, ranging from deep seas, coral reefs, and coastal and estuarine waters to rivers and lakes. Several barriers have blocked aquatic animals from radiating, among which the interface between saline and freshwater habitats is one of the most formidable barriers. Only limited groups have overcome such boundaries Although freshwater occupies a small portion of Earth's surface (0.8%) Takifugu rubripes and Tetraodon nigroviridis, were proposed as model systems for the evolution of vertebrate genomes The Tetraodontidae, known as pufferfishes, are highly derived euteleosts. Composed of 189 species placed in 19 genera To provide an overview of the evolutionary history of freshwater tetraodontids, we used whole mitogenome sequences from 45 tetraodontids (including 31 newly determined sequences) plus those from five outgroups. Unambiguously aligned sequences were subjected to partitioned maximum likelihood (ML) analysis and divergence time estimation. Our results show that tetraodontids entered freshwaters in the different continents during at least three different times and that the timing of these habitat shifts corresponded with geological events in each continent.Complete L-strand nucleotide sequences from the mitogenomes of the 31 species examined were deposited in the DNA DataBank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), and GenBank . The genBoth the pairwise transition (TS) and transversion (TV) differences for each partition increase with increasing evolutionary distance. The exceptions are TS differences at the third codon positions in the protein-coding genes , in whicn3rRTn) was expected to effectively remove the noise expected from quickly saturated transitional changes in the third codon positions and avoid a lack of signal by retaining all available positions in the dataset n3rRTn dataset is shown in Based on the patterns of sequence variation, the dataset with the third codon positions converted by RY-coding  . TetraodColomesus asellus) is deeply nested in Clade B together with a congeneric, euryhaline species (C. psittacus). Central African and Southeast Asian freshwater species fall into two different clades that are nested within Clade D , with a sister-group relationship with Chelonodon species (BP = 100%)  . Southea = 100%) .Tetraodon, Torquigener, and Sphoeroides, were found as non-monophyletic, and monophyly of each genus was confidently rejected by the approximately unbiased (AU) test (P = 0.000).Three genera, Colomesus asellus, diverged from C. psittacus at 10 MA (4–18 MA) in Clade B (Chelondon species at 38 MA (21–59 MA), and their most recent common ancestor emerged at 17 MA (6–31 MA)  , Table 2P0 = 0.998); this habitat is found in all four major lineages in the family and is also dominant among both extant members and ancestral nodes (P1 = 0.980) and Southeast Asian (P1 = 0.945) freshwater clades  in tetraodontid pufferfishes is shown in al nodes . The opeal nodes . The frer clades . Accordir clades .Canthigaster) and Tetraodontinae P = 0.000), as also reported in previous molecular studies Tetraodontid pufferfishes have long attracted biologists across diverse fields, such as morphology, toxicology, and molecular biology, but their phylogenetic relationships have remained unexplored. A morphology-based classification that divides this family into two subfamilies, Canthigasterinae  test (P = 0.000). Phylogenies of these genera have never been investigated by either morphology or molecules and generic classification should be revised in future studies.Three genera, Our results for the timing of divergences are largely congruent with previous mitogenomic studies Mitogenomic hypotheses on teleostean timescales also show strong discrepancies from fossil evidence P = 0.000). The ML reconstruction of ancestral habitats demonstrates that the tetraodontids independently invaded freshwater habitats in the different continents at least three separate times , which are often mistakenly regarded as freshwater pufferfishes, have considerable resistance to freshwater exposure and are widely sold in the freshwater aquarium trade Most coastal pufferfishes are reported to temporarily occur in brackish and freshwater regions for rest or refuge Colomesus asellus is widely distributed in the Amazon River basin from Peru to Marajó Island, including tributaries of the Araguaia and Guaporé rivers, the lower reaches of the Orinoco River basin, and the Essequibo River basin C. psittacus occurs in brackish and coastal waters around the north of South America Colomesus species indicate that their ancestors invaded the western Amazonian through the latter Miocene marine incursion (11–12 MA) and subsequently diverged from the marine and brackish lineage with adaptation to freshwater habitats.Tetraodon exclusively inhabit freshwater environments. Tetraodon duboisi, T. mbu, T. miurus, and T. schoutedeni are distributed in the Congo basin, T. pastulatus is found in the Cross River in the southeastern Nigerian delta, and T. lineatus has a much wider occurrence, ranging from the Senegal and Volta basins to the Niger and Nile basins and Lake Turcana Tetraodon species occurred between 17–38 MA , Auriglobus spp. , Tetraodon cutcutia (East India to Myanmar), and other Tetraodon species  Southeast Asian freshwater pufferfishes include approximately 21 species in three to five genera, which are distributed from Southwest India to Indochina and the Sunda Islands. They have speciated considerably in Indochina and the Sunda Islands, particularly in the Mekong Basin, while Phylogenetic analysis of whole mitogenomic datasets reveals that tetraodontid species that spend all of their life stages in freshwater in South America, Central Africa, and Southeast Asia are independently nested in two of the four lineages, and the ML reconstruction of ancestral habitat states demonstrates that tetraodontids independently invaded freshwater habitats in the different continents at least three separate times. The relaxed molecular clock analysis of divergence times showed that the timings of these invasions were different for each continent, occurring around 0–10 MA in South America, 17–38 MA in Central Africa, and 48–78 MA in Southeast Asia. These timings are congruent with geological events, such as marine incursions, that could facilitate adaptation to freshwaters. Although many pufferfishes have some resistance to freshwater exposure, invasions into freshwater habitats occurred rarely, not repetitively. This suggests the difficulties faced by marine pufferfishes when invading occupied niches in freshwater environments.Of the currently recognized 28 freshwater species , Mola mola and Ranzania laevis (Molidae), and Diodon holocanthus and Chilomycterus reticulatus (Diodontidae), based on the results of Yamanoue et al. We tried to choose at least two species for each genera, except for monotypic genera; however, only one species was available for several genera, including A portion of the epaxial musculature or fin clips (∼0.25 g) was excised from fresh specimens of each species and immediately preserved in 99.5% ethanol. Total genomic DNA was extracted using a Qiagen DNeasy tissue kit (Qiagen) following the manufacturer's protocol. The mitogenomes were amplified in their entirety using a long PCR technique A total of 181 fish-versatile PCR primers were used in various combinations to amplify the contiguous, overlapping segments of the entire mitogenome, and 20 species-specific primers were designed when no appropriate primers were available. A list of the PCR primers used in this study is available from Y.Y. upon request.Long PCR and subsequent short PCR were performed as previously described The DNA sequences were edited and analyzed with EditView v.1.0.1, AutoAssembler v.2.1 (Applied Biosystems) and DNASIS v.3.2 (Hitachi Software Engineering). A total of 13 protein-coding, 22 tRNA, and two rRNA gene sequences for 31 species were aligned using ProAlign v.0.5 nRTn, where n denotes nucleotides) and 2) the third codon positions converted to purine (R) and pyrimidine (Y)  to remove the effects of saturation on branch length estimation. The aligned sequence data in NEXUS format are available from Y.Y. upon request.We constructed two different datasets to investigate the effects of quickly saturating the third codon positions in the protein-coding genes on the phylogeny estimation: 1) all aligned positions of gene-coding regions of mitogenomic sequences  across genes than across codon positions (or types of molecule) within genes, at least for a set of mitochondrial genes. ModelTest v.3.0.6 nRTn dataset. The ML analysis was conducted with RAxML v.7.2.6 The aligned datasets were subjected to an ML analysis. We set five partitions for the unambiguously aligned nucleotide sequences of the 123n3rRTn dataset using the likelihood-based approximately unbiased (AU) test P<0.05 was considered significantly different.Alternative tree topologies were individually compared to the resulting ML tree from the 12n3rRTn dataset was used for divergence time estimation, and ML estimates of branch lengths were obtained using the BASEML program in PAML under the GTR+Γ substitution model with the gamma prior set at 0.2. Two priors, the overall substitution rate (rgene gamma) and the rate-drift parameter (sigma2 gamma), were set at G  and G , respectively, using the strict molecular-clock assumption with a 75 MA constraint  model λ = μ = 1 and ρ = 0, so that the priors were similar to those used in previous mitogenomic studies using MULTIDIVTIME To investigate the relative timings of major cladogenetic events in the family Tetraodontidae, a relaxed molecular-clock Bayesian method implemented in MCMCTREE in PAML v.4.4 Tetraodon nigroviridis and Takifugu rubripes, and between Tetraodontiformes and its sister group Caproidei, at 73 MA (with a 95% CI of 57–94 MA) and 160 MA (with a 95% CI of 133–194 MA), respectively, using whole mitogenome sequences and multiple fossil calibration points with a partitioned Bayesian method Takifugu and Tetraodon (Sphoeroides from the Pliocene (5 MA) The MCMCTREE program allows for minimum (lower) and maximum (upper) time constraints, and it has been argued that multiple calibration points provide more realistic divergence time estimates overall etraodon . Moreovep = 0.1 and c = 1 as the default http://tree.bio.ed.ac.uk/software/tracer/) to check mixing, choose a suitable burn-in, and look for trends that might suggest problems with convergence. The number of samples  was large enough to reach effective sample sizes (ESSs>200) for all parameters estimated in this study.A softbound version of the program (MCMCTREE) was used, so that probabilities of the true divergence time that fell outside the maximum and minimum bounds were not zero n3rRTn dataset with branch lengths fixed by MCMCTREE, and the likelihood scores of these four character states were computed for each node of the tree.Habitats of tetraodontid pufferfishes were variable, ranging from freshwater, brackish and estuarine water, and coastal water, to the bottom of the continental shelf and open sea. In this analysis, their habitats were simply classified into four categories for convenience. First, species that spend all of their life stages in freshwater were categorized as having a freshwater habitat. The ecology of tetraodontid pufferfishes that inhabit coastal waters varies greatly with species. Some spend part of their life in brackish and freshwater regions, while others rarely enter freshwater Figure S1Node numbers on the best-scoring ML tree for showing divergence time estimates in (TIF)Click here for additional data file.Table S1Summary of MCMC samples for node ages (MA) in the independent-rates analyses using MCMCTREE. For node numbers, see (DOC)Click here for additional data file.


Pulmonary embolism may escape prompt diagnosis since clinical symptoms and signs are nonspecific. The occurrence of syncope as the sole initial symptom in a previously healthy patient with no predisposing factors to embolism and no hemodynamic instability is extremely rare, which may have been a factor in the delayed diagnosis. We describe a case of agnogenic massive pulmonary embolism with syncope as the initial symptom. A 41-year-old previously healthy female was admitted to the Department of Neurology, Taizhou People’s Hospital in March 2012, for two transitory episodes of syncope during a 5-h period. Following admission, chest computed tomography demonstrated embolism in the right main pulmonary and left inferior pulmonary arteries. Color ultrasonography revealed a dilated right ventricle and right heart overload, severe tricuspid regurgitation and severe pulmonary hypertension. Following the final diagnosis, the patient was successfully treated with interventional mechanical thrombectomy combined with thrombolytic therapy with local and systemic low-dose urokinase. We consider that raised awareness and early diagnosis and treatment were key factors in ensuring a satisfactory prognosis. Acute massive pulmonary embolism (MPE) is a critical disease associated with high mortality in clinical practice. Approximately 79% of patients have coincident deep vein thrombosis . ClinicaA 41-year-old previously healthy female was admitted to the Department of Neurology, Taizhou People’s Hospital in March 2012, for two transitory episodes of syncope during a 5-h period. The patient had an unhealthy lifestyle of physical inactivity. No urinary and fecal incontinence, general fatigue, chest pain, breathing difficulty, hemoptysis or fever were observed during the course of disease. Following admission, physical examinations revealed a body weight of 75 kg, body height of 159 cm, body temperature of 36.0°C, pulse of 80 bpm, respiratory rate of 23 bpm and blood pressure of 120/60 mmHg. The patient had a slightly haggard expression, no cyanosis of the lips and no jugular vein distention. Bilateral respiratory movements were identical and vocal fremitus was equal. Dullness was heard in the right lower lung on percussion. Breath sounds were diminished and no moist rales were heard. The patient’s heart rhythm was regular, P2>A2 (pulmonary second sound was higher than aortic second heart sound) and there was no edema in the lower extremities.9 cells/l and the percentage of large white blood cells was 57.2%. Biochemical tests revealed 1.4 mmol/l triglycerides, 0.75 mmol/l high-density lipoprotein and 3.61 mmol/l low-density lipoprotein. Blood gas analysis revealed a pH of 7.471, 61.1 mmHg PaO2, 23.5 mmHg PaCO2 and 18.2 mmol/l HCO3− . Chest radiographs revealed pulmonary hilar enlargement and a broadened shadow on the right superior pulmonary artery. An electrocardiogram revealed a flat V1–V3 T wave and magnetic resonance angiography of the head revealed ∼60% luminal stenoses of the right posterior cerebral artery and the left external carotid artery.A complete blood test revealed a white cell count of 11.21×10μg qd) to boost the cerebral circulation, without effect. A further episode of syncope occurred during the 18 h after admission and the patient was transferred to the Department of Respiratory Medicine for a D-dimer assay, which indicated a value of 1,200 μg/l. An enhanced chest computed tomography (CT) scan revealed filling defects in the right main pulmonary and left inferior pulmonary arteries, as well as bilateral pleural effusion  and intravenous Alprostadil for injection .Following the above therapies, digital subtraction angiography (DSA) of the deep veins of the lower limbs and the inferior vena cava demonstrated unobstructed blood flow, with no apparent thrombosis. A 4–5F double J tube was inserted through the right femoral vein to the main pulmonary artery for DSA of the pulmonary artery, to confirm the filling defect in the right main pulmonary artery . An exchA postoperative intravenous drip of low-dose urokinase  was initiated and the patient was also treated with 5,000 units low-molecular-weight heparin, administered subcutaneously once every 12 h for three consecutive days. Warfarin (2.5 mg) was administered orally once every 12 h on a daily basis. The prothrombin time (PT) and international normalized ratio (INR) were monitored; when the PT and INR were twice and 2.5 times their respective normal levels, warfarin therapy was administered singly, plus anti-infective, supportive and oxygen therapies.Three days after surgery, the patient demonstrated a distinctly improved mental condition with no further syncopal attacks. An enhanced chest CT scan 10 days after surgery revealed evident improvement of the thrombosis in the right main pulmonary artery and left inferior pulmonary artery branch, as well as disappearance of the pleural effusion, compared with the previous chest CT scan . Color uMPE refers to an embolism of the main pulmonary trunk and is associated with high mortality ,5. The mSyncope as the sole initial symptom has been reported in MPE patients with comorbid hemodynamic disturbances ,8,9. TheMPE combined with hemodynamic disturbance is an absolute indication of thrombolysis; although thrombolytic therapy via a peripheral vein has a delayed effect and a high risk of hemorrhage. Certain patients are unable to benefit from this treatment due to contraindications to thrombolysis. Surgical thrombectomy of the pulmonary artery is also associated with high mortality and disability risks . HoweverCompared with venous thrombolysis, local pulmonary artery thrombolysis via a catheter elevates the focal drug concentration and reduces the required dosage of thrombolytic medications, thus enhancing the therapeutic effects and reducing the incidence of hemorrhagic complications. The procedure of mechanical embolus disintegration and thrombectomy by a catheter permits the clearance of embolus fragments and entry into the distal pulmonary artery, realizes the unblocking of the clogged central pulmonary artery, improves pulmonary perfusion, reduces pulmonary artery pressure and improves right ventricular function, consequently resulting in an increased clinical treatment success rate.In conclusion, the occurrence of syncope as the sole initial symptom in a previously healthy patient with no predisposing factors to embolism and no hemodynamic instability following admission is extremely rare, which may have been a factor in the delayed diagnosis of pulmonary thromboembolism. In this situation, the raised awareness of diagnosis and knowledge concerning the clinical presentation of pulmonary thromboembolism are key factors in ensuring an immediate diagnosis and adequate intervention.


WFS1 gene, which lead to endoplasmic reticulum (ER) stress-mediated cell death. Case studies have found widespread brain atrophy in late stage WFS. However, it is not known when in the disease course these brain abnormalities arise, and whether there is differential vulnerability across brain regions and tissue classes. To address this limitation, we quantified regional brain abnormalities across multiple imaging modalities in a cohort of young patients in relatively early stages of WFS. Children and young adults with WFS were evaluated with neurological, cognitive and structural magnetic resonance imaging measures. Compared to normative data, the WFS group had intact cognition, significant anxiety and depression, and gait abnormalities. Compared to healthy and type 1 diabetic control groups, the WFS group had smaller intracranial volume and preferentially affected gray matter volume and white matter microstructural integrity in the brainstem, cerebellum and optic radiations. Abnormalities were detected in even the youngest patients with mildest symptoms, and some measures did not follow the typical age-dependent developmental trajectory. These results establish that WFS is associated with smaller intracranial volume with specific abnormalities in the brainstem and cerebellum, even at the earliest stage of clinical symptoms. This pattern of abnormalities suggests that WFS has a pronounced impact on early brain development in addition to later neurodegenerative effects, representing a significant new insight into the WFS disease process. Longitudinal studies will be critical for confirming and expanding our understanding of the impact of ER stress dysregulation on brain development.Wolfram Syndrome (WFS) is a rare autosomal recessive disease characterized by insulin-dependent diabetes mellitus, optic nerve atrophy, diabetes insipidus, deafness, and neurological dysfunction leading to death in mid-adulthood. WFS is caused by mutations in the  WFS1) was identified by our group in 1998 WFS1 encodes an endoplasmic reticulum (ER) membrane-embedded protein called wolframin Wolfram syndrome (WFS) is a rare  autosomal recessive genetic disease characterized by early childhood onset insulin dependent diabetes, optic nerve atrophy, vision and hearing loss, diabetes insipidus and neurodegeneration, resulting in death in middle adulthood, typically due to brainstem atrophy-induced respiratory failure Although there are no current treatments for this disease, agents to treat ER stress-mediated apoptosis are currently in development Our current knowledge of WFS-related brain changes come primarily from clinical exams and post-mortem neuropathological case studies of severely affected individuals with WFS. These studies found abnormalities in multiple regions of the brain, including hypothalamus, pituitary, pons, inferior olivary nucleus, lateral geniculate nucleus, thalamus, optic nerve, optic tract and cerebellum in vivo structural brain imaging techniques and neurobehavioral measurements.An understanding of the natural history of neurological changes in WFS will be important in assessing the validity of animal models of WFS, for developing interventions for neurological changes, as well as addressing questions about how WFS1 mutations and ER stress affects neurodevelopmental processes. Thus, the goal of the current study is to determine the regional pattern of neuroanatomical abnormalities in WFS across a range of disease duration and imaging modalities. To address this gap in the literature, we studied individuals with WFS at the earliest stage of disease possible and compared them to both age and gender equivalent healthy and type 1 diabetic controls. Thus, we were able to survey the brain’s structural and functional integrity across a range of very early to more advanced disease using sophisticated Participants with WFS were evaluated clinically by a team of investigators including a pediatric neurologist, audiologist and ophthalmologist. Patients then performed neuropsychological testing and MRI scans and completed self-report questionnaires if over 16 (parents completed parent-report if 16 or younger) about psychiatric and behavioral issues. Control subjects completed MRI scans only.http://wolframsyndrome.dom.wustl.edu/medical-research/Wolfram-Syndrome-Home.aspx), who were under the age of 30, aware of their diagnosis and able to travel were invited to participate. Further inclusion criteria were the diagnosis of diabetes mellitus and optic nerve atrophy before 18 years of age or genetic confirmation of a WFS1 mutation.Individuals with WFS who registered on the Washington University WFS Registry  and individuals with type 1 diabetes mellitus (Type 1 Controls or T1C) scanned at Washington University as part of other studies. Youth with T1C were recruited from the Pediatric Diabetes Clinic at St. Louis Children’s Hospital and Washington University School of Medicine in St. Louis. Non-diabetic controls were either healthy siblings of the diabetic patients or from the community. T1C were excluded for diagnosed psychiatric disorder, significant neurological history not due to diabetes, known premature birth (<36 weeks gestation) with complications, psychoactive medications, or physical limitations that would interfere with testing. No T1C participants had known retinopathy, nephropathy or neuropathy at the time of testing. Controls were excluded for current or past history of neurological and psychiatric diagnoses or other significant health conditions. All subjects underwent MRI scans for research purposes only, not for any clinical indication.For all participants, informed consent was obtained prior to participation and the study was approved by the Human Research Protection Office at Washington University in St. Louis. Children under age 18 provided assent, and their parent/guardian provided written consent.Before beginning testing, WFS participants were determined to have blood glucose levels above 70 mg/dl. Standardized IQ, cognitive and behavioral measures were administered. Each patient’s performance was compared to normative data for their age and gender.Vocabulary and Similarities subtests from The Wechsler Abbreviated Scale of Intelligence (WASI) Conners’ Continuous Performance Test II (CPT II)Letter Number SequencingChildren’s or Adult California Verbal Learning Test (CVLT)Achenbach Child Behavior Checklist or Adult Self Report  Before beginning scanning, WFS and T1C participants were determined to have blood glucose levels above 70 mg/dl. Scans were acquired on one of three Siemens 3T Tim Trio MRI scanners at Washington University. All three scanners had the same hardware and software, and they are each checked by Siemens maintenance personnel every 3 months to confirm that the machines are running in accordance with their recommendations. In addition, cross-calibration scanning on 15 normal control children from another study (unpublished data) using similar sequences as ours has shown a high degree of consistency and absolute difference in regional DTI FA  and Freesurfer-derived volumes . These metrics are consistent with the literature of Freesurfer performance across different scanners T1-weighted MPRAGE sequence: Sagittal acquisition, TR = 2400, TE = 3.16, TI = 1000, voxel resolution = 1×1×1mm, Time = 8:09 min. T2-weighted MR: Sagittal acquisition, TR = 3200, TE = 455, voxel resolution = 1×1×1mm, Time = 4:43 min. T2 FLAIR: Transverse acquisition, TR = 9190, TE = 98, TI = 2500, voxel resolution = .86×.86×3mm, Time = 3:59 min. This sequence was acquired for the WFS group only for clinical neuroradiological reading. Diffusion Tensor Imaging (DTI): The echo planar sequence consisted of 27 directions with the b-values ranging from 0 to 1400 s/mm2. Transverse acquisition, TR = 12300, TE = 108, voxel resolution = 1.98×1.98×2 mm, Time = 5:44 min. The DTI sequences in the control groups varied slightly in the number of b = 0 scans acquired, but their scans had the same resolution and range of b-values as the WFS group .The following sequences were acquired: Methods were selected to provide complementary levels of information. The ROI method has several advantages over voxel-wise analyses – it minimizes the risk of type 1 error, avoids the many statistical assumptions of voxel-wise analyses, and allows for tailoring ROIs to avoid partial volume effects, thus reducing the impact of imperfect registration procedures To determine regional gray and white matter brain volumes from anatomically defined regions, we used the semi-automatic segmentation program Freesurfer (v5.0) To examine the brainstem, known to be particularly affected in advanced WFS, in more detail, we performed manual segmentation of the Freesurfer-generated brainstem according to published criteria To explore cortical metrics in a landmark-independent manner, we reconstructed and segmented individual subjects’ cortical surfaces using Freesurfer (v5.0) www.fil.ion.ucl.ac.uk). Images were simultaneously normalized to Montreal Neurological Institute (MNI) space using non-linear transforms, corrected for intensity inhomogeneity, and segmented into gray matter, white matter, and CSF. We used custom tissue probability maps based on our subjects instead of the default SPM maps to improve segmentation Voxel-based morphometry (VBM) was performed with statistical parametric mapping software  were computed Regions were selected based on a well-established DTI atlas Despite the advantages of the ROI method of DTI, we also used voxel-wise exploratory analyses of our DTI data to confirm our ROI findings but also to be open to white matter effects in other locations without regard for anatomical boundaries. Diffusion weighted images were internally motion corrected using 9-parameter affine (rigid body + scanner axis stretch) registration using in-house developed software. Then, using each individual’s motion-corrected, aligned, and averaged DWI data set, BET (FMRIB Brain Extraction Tool) was used to compute a brain mask, and FDT (FMRIB diffusion toolbox) was used to compute FA maps. To create a target template for registration, a subset of individuals were chosen from the entire subject pool to provide an even distribution of males and females and ages. FA images in the target group were then nonlinearly aligned to 1×1×1 mm space and to each other. The target image was the image that requires the least amount of warping for all other FA images to align to it. All FA images in the entire subject pool were then nonlinearly aligned to each other in 1×1×1 mm space and to the target image. The target image was affine-aligned into MNI152 standard space, and every FA image was then transformed into 1×1×1 mm MNI152 space. A mean FA image was calculated and used to produce the mean FA skeleton to represent the center of white matter tracts. FA images were projected onto the mean FA skeleton and thresholded at FA = 0.2 for voxel-wise analyses. Mean, axial and radial diffusivity images were also calculated and all were analyzed with Randomise, a permutation-based multiple comparisons corrected statistical approach For all region-based analyses we performed univariate analyses of each region, with group as the independent variable  with age and gender covaried. We were then able to determine if control groups differed on regional values. We applied a Bonferroni correction for multiple comparisons within imaging modalities. For gray and white matter volumes of interest, the criterion for significance was p<.0038 (.05/13). For DTI regions of interest, the criterion for significance was p<.005 (.05/10). Regions that survived correction from any analyses were examined for differences between groups with post-hoc comparisons. For voxel or vertex-wise analyses, we compared all WFS vs all controls, to simplify the statistical models needed  and to maximize power. Each analysis used software-specific multiple comparison correction procedures described above, and age and gender were removed as covariates from all models.WFS1 gene and had insulin dependent diabetes mellitus . All but one had been diagnosed with optic nerve atrophy at the time of assessment ; 10/14 were being treated for diabetes insipidus . WFS individuals had a wide range of duration of diabetes mellitus , optic atrophy  and diabetes insipidus . Younger individuals tended to have shorter duration of all of these symptoms. Standard ophthalmological tests revealed that 13/14 WFS participants had color vision impairment ranging from mild to severe. All had abnormally thin retinal nerve fiber layers on optical coherence tomography (OCT). Audiometric tests revealed that 7/14 patients had evidence of hearing loss. Clinical neurologic exams revealed that the most common abnormalities were horizontal nystagmus and difficulty tandem walking (8/14 subjects). Several subjects (5/14) had impaired vibration sense, and other more subtle motor abnormalities were also noted, including increased lower extremity tone, intention tremor, and brisk deep tendon reflexes in one subject each. These clinical findings confirm that our WFS group ranged from very mild to moderate severity of symptoms on standard clinical measures.Patients with WFS ranged in age from 5.9 to 25.8 . All had confirmed mutations on the Some individuals with WFS, either due to language  or sensory issues , could not complete all of the tasks. However, the rest of the WFS group performed above the average range for verbal intelligence , and within the average range for verbal learning and memory , and attention . In contrast, on a comprehensive psychological questionnaire, participants or parents reported more symptoms in the Internalizing Domain which includes depression, anxiety and somatic symptoms  than in the Externalizing Domain which includes conduct disorders, impulsivity and aggression  = 3.85, p = .002). Five individuals had clinically significant symptoms (T score above 69) and 1 had clinically borderline symptoms (T score 65–69) in the Internalizing domain, but only 1 had clinically significant symptoms in the Externalizing Domain. Eleven WFS subjects had blood glucose levels recorded at the time of testing; levels ranged from 124 to 325 .MRIs were acquired on 11 individuals with WFS. The remaining 3 patients had contraindications for MR and could not be safely scanned . Two of the 11 had clinically normal scans; the other 9 had clinically detectable abnormalities on MR similar to those noted in case studies of WFS  and individuals with diabetes insipidus .De-identified scans from age and gender-equivalent HC and T1C individuals were obtained for comparisons to the WFS group. These subjects either had T1-weighted images  or DTI scans . Thirty-eight subjects had T1-weighted scans and 12 had DTI scans on the first scanner, 25 (including the 11 WFS subjects) had T1-weighted scans and 38 (including the 11 WFS) had DTI scans on the second scanner and no subjects had T1-weighted scans, and 16 had DTI scans on the third scanner. Within DTI sequences, 50 subjects had b1400 and 16 had b1000 scans; 42 had 25 directions and 24 had 26 direction scans. The control groups were not different in mean age or gender distribution from the WFS group . In bothOverall, there was a significant effect of group  on intracranial volume (ICV) after correcting for age and gender  = 5.9, p = .005). Post-hoc pair-wise comparisons revealed that the WFS group had smaller ICV compared to the HC and T1C groups (p<.004), but these control groups were not different from each other (p = .91). Whole brain volume  was also smaller in the WFS group  = 5.1, p = .009), but after controlling for ICV, was similar across groups  = 0.9, p = .38).Out of 13 regions normalized for ICV, 5 had a main effect of group at the p<.05 level; 3 of these survived multiple comparison correction (p<.0038); Fig 1. Volumes of brainstem segments  were normalized for ICV. A repeated measures general linear model analysis found, as expected, a main effect of diagnosis  = 22.4, p<.001; WFS < T1C and HC) and a main effect of segment  = 41.3, p<.001; pons > midbrain and medulla) with age and gender as covariates. More interestingly, we found a segmental volume by diagnosis interaction  = 7.6, p<.001) that was still significant after also covarying duration of diabetes  = 9.1, p<.001). Post-hoc contrasts found that the WFS group volume was significantly different from the control groups for all three segments, but the effect was most striking in the pons . Before 2, cluster-wise p  = .0008), a lingual region , and two rostral middle frontal regions . In the right hemisphere, there was also reduced thickness in the WFS group in a rostral middle frontal region . No clusters from the surface analyses of surface area or volume had significant group effects after multiple comparison correction.We examined cortical thickness, cortical area and cortical volume (a product of thickness and area) between the combined control group and the WFS group across the entire cortex. In vertex-wise analyses, the WFS group had reduced thickness in several regions after controlling for age and gender, and applying multiple comparison corrections . In the VBM analyses of gray matter revealed that the WFS had significantly less gray matter volume in the cerebellum. Two large clusters survived multiple comparison and smoothness correction, covarying for age and gender: one in the right cerebellum  and one in the left cerebellum . There wPlaced regions were visually verified to not overlap with any of the T2 signal abnormalities observed in WFS patients. Out of the 10 regions examined for DTI measures, 3 had a main effect of group for FA and 3 had a main effect of group for MD measures at the p<.05 level. Two FA regions  = 13.7, p<.001; optic radiation: F = 11.0, p<.001) and one MD region  = 14.0, p<.001) survived multiple comparison correction with age and gender as covariates  . For FA Significant reduction in FA was found in the WFS group across a wide range of regions after controlling for age and gender and correcting for multiple comparisons. The largest and most striking clusters were found in the brainstem, cerebellum and optic radiations see , consistTo assess any impact of differences in scanners, we performed further analyses on the significant regions, restricting analyses to only those subjects with data collected on the same 3T scanner. For subcortical volumes  and DTI measures  analyses revealed the same pattern as our larger sample . These results survive multiple comparison correction and support the robustness of our findings, suggesting that scanner and sequence variability within our control groups did not bias the original results.We report quantitatively measured and regionally distinct structural brain changes in early stage WFS. We found reduced ICV, dramatically reduced regional volumes and altered white matter microstructural integrity. The brainstem, cerebellum and optic radiations were particularly affected in WFS compared to both healthy controls and controls with type 1 diabetes (T1C). Notably, decreased brainstem and cerebellum volumes  were seen in almost all individuals with WFS, regardless of age or duration of diabetes or other features of the disease. These data together suggest that WFS affects brain development both globally and preferentially, even at the earliest stages of symptoms. Previous clinical retrospective survey studies suggested that neurological features occur later (15–30 yrs of age) in the disease process Our observation that individuals with WFS have significantly reduced ICV, in comparison to both diabetics and healthy controls, supports the concept of aberrant neurodevelopment in WFS. The major expansion of the cranial vault is driven by brain growth early in childhood until all of the cranial sutures fuse (around age 6), at which point ICV changes only very slowly The preferential vulnerability of the brainstem and cerebellum in WFS has been described previously, generally in adults in advanced disease states, without quantification or control groups In addition to these profound subcortical and cerebellar changes, we also found relatively isolated cortical effects of WFS. Cortical thickness was reduced in WFS in restricted regions, including lingual, precentral, and rostral middle frontal cortex. These cortical regions are associated with vision, motor function and higher order cortical functions such as working memory, respectively. These regions are known to reduce in thickness with age during middle childhood, and be influenced by genetic factors Our complementary, quantitative measurements of structural brain differences in WFS allow us to build hypotheses about potential underlying mechanisms. For example, we found reduced fractional anisotropy and reduced axial  diffusivity in the brainstem, cerebellum, optic radiations and numerous other regions. Radial diffusivity, the diffusion of water perpendicular to white matter fibers, is known to increase in response to myelin damage While WFS has previously been considered a neurodegenerative disease, it is also possible that the brain abnormalities reported in WFS are the product of two separate pathological processes, one that is neurodevelopmental in nature and the other neurodegenerative. Another possibility is that although there are early brain changes, these do not produce neurological symptoms until later in development, due to compensatory processes or the fact that the involved brain networks and functions do not come on-line until later Our cohort also had notable depression and anxiety, similar to findings in advanced WFS The major strength of this study is its quantification of regional brain abnormalities in a group of WFS individuals with a range of disease severity, from newly diagnosed to more significantly affected. Although the WFS sample presented here is small compared to studies of more common neurodegenerative diseases, it is the largest quantified neuroimaging study of WFS to date. Furthermore, we compare our WFS to two larger groups of age and gender equivalent comparison groups, to control for any effects of diabetes or normal development on the brain, and we applied multiple comparison corrections. However, there are some limitations to our study. Since our study design was cross-sectional, we cannot be sure that the observed age-independence of certain effects would be confirmed in a longitudinal within-subjects design. Control subjects were a convenience sample obtained from several current studies at WUSM, and thus did not have cognitive/behavioral testing. Although all WFS participants who were willing to travel to St. Louis were accepted into the study, this requirement may have selected for individuals who were higher functioning than the general WFS population. The T1C subjects with DTI scans had shorter duration of diabetes than our WFS subjects. Ideally, groups would be matched on this variable and on degree of hyperglycemia and hypoglycemia exposure. Based on our experience and the existing literature, however, we feel that it is highly unlikely that small differences in duration of diabetes would cause the dramatic differences seen between WFS and the control groups . In all of the previous neuroimaging studies of T1C, including those in youth The results of this study lay the groundwork for larger, longitudinal investigations of neuroanatomical changes in WFS which could confirm and extend our observations, and distinguish between earlier vs. later occurring abnormalities. In addition, the behavioral correlates of neuroanatomical changes will need to be better understood. For example, functions known to be reliant on the optimal development of the cerebellum and brainstem, such as gait and balance, should be better characterized. Longitudinal measurement of this cohort of individuals with WFS is ongoing and will need to be significantly expanded to answer these important clinical questions. Nevertheless, our current data provide fundamental insight into the neurophenotype associated with WFS and provide the necessary information for determining the appropriateness of animal models of WFS for understanding the neurobiological mechanisms underlying these findings and for developing neurologically-targeted interventions. This work would also contribute to our understanding of the impact of ER stress-related dysfunction during development.


Canis lupus) movements anywhere near the northern extreme of the species' range in the High Arctic (>75°N latitude) are lacking. There, wolves prey primarily on muskoxen (Ovibos moschatus) and must survive 4 months of 24 hr/day winter darkness and temperatures reaching −53 C. The extent to which wolves remain active and prey on muskoxen during the dark period are unknown, for the closest area where information is available about winter wolf movements is >2,250 km south. We studied a pack of ≥20 wolves on Ellesmere Island, Nunavut, Canada (80°N latitude) from July 2009 through mid-April 2010 by collaring a lead wolf with a Global Positioning System (GPS)/Argos radio collar. The collar recorded the wolf's precise locations at 6:00 a.m. and 6:00 p.m. daily and transmitted the locations by satellite to our email. Straight-line distances between consecutive 12-hr locations varied between 0 and 76 km. Mean (SE) linear distance between consecutive locations (n = 554) was 11 (0.5) km. Total minimum distance traveled was 5,979 km, and total area covered was 6,640 km2, the largest wolf range reported. The wolf and presumably his pack once made a 263-km (straight-line distance) foray to the southeast during 19–28 January 2010, returning 29 January to 1 February at an average of 41 km/day straight-line distances between 12-hr locations. This study produced the first detailed movement information about any large mammal in the High Arctic, and the average movements during the dark period did not differ from those afterwards. Wolf movements during the dark period in the highest latitudes match those of the other seasons and generally those of wolves in lower latitudes, and, at least with the gross movements measurable by our methods, the 4-month period without direct sunlight produced little change in movements.Information about wolf ( Canis lupus) movements have been well studied throughout much of the species' circumpolar range Animal movements, daily, seasonal, and annual, reveal a great deal of information about a species and its natural history Ovibos moschatus) However, information about wolf movements near the northern extreme of the species' range in the High Arctic (>75°N latitude) are lacking, and that area is of interest to study because that is the only region at which wolf movements during winter must take place during 4 months of 24 hr/day darkness and in temperatures as low as −53 C We studied the movements of a pack of ≥20 wolves at 80°N latitude for 9 months including the 4-month dark period in an area where their main prey was muskoxen.Lepus arcticus) have long been common in the area Rangifer tarandus pearyi), and wolves have denned there for decades or even centuries We conducted this study primarily on the Fosheim Peninsula of Ellesmere Island near Eureka , Nunavut, Canada. A weather station is located at Eureka in the east central part of the Fosheim Peninsula north of Slidre Fiord. The amount of daylight in the area varies from 24 hours per day from 16 April to 26 August to 24 hours of darkness per day from 18 October to 23 February. Mean daily temperatures vary from 5.7 C in July to −38.4 C in February. Wolves, muskoxen, and arctic hares , and estimated home range with the Animal Movement extension The collared wolf visited the vicinity of the Eureka Weather Station 16 times between 7 August 2009 and 16 April 2010, and seven times personnel there counted the wolves, photographed them, and emailed us the photos . When thWe used program R The collared wolf was with a group of four other wolves when we darted him and had just been dominating one of them 2 between 9 July 2009 and 18 October 2009 on the Fosheim Peninsula of Ellesmere Island  linear distance between consecutive locations was 11 km , and mean (SE) linear distance between consecutive locations that were >500-m apart was 12 km . Total minimum distance traveled was 5,979 km. The collared wolf, and presumably his pack, covered an area of 1,642 kme Island . They thsland  foray to the southeast during 19–29 January 2010, returning from 29 January to 1 February. During the return trip, the animal covered an average of 41 km/day straight-line distances between consecutive 12-hour locations. We have no direct evidence about whether the collared wolf was accompanied by the rest of the pack. However, patterns of consecutive locations later determined to indicate prey carcasses visited (>2 consecutive 12-hr locations the same) were no different on this foray than during movements before and after when the full pack was travelling together. We believe this behavior indicates that the pack accompanied the collared wolf.P≤0.05) except that the mean for September–October was significantly less than those for November–December and January–February . Our collared wolf would have been an estimated 10-years old at death, uncommonly old for a wild wolf The collared wolf died about 12 April 2010. The animal was emaciated and had an enlarged spleen. Some 75% of dogs with enlarged spleens suffer from the cancer hemangiosarcoma, which can cause, among other symptoms, loss of appetite (2 over a period of 8.5 months, several times larger than that of any other known wolf pack Our study pack of ≥20 wolves covered an area of 6,640 kmOur study pack traveled throughout winter and the 4-months of 24-hr/day period of darkness at distances similar to those before and after this period. That is, the animals maintained their regular travel schedule throughout the dark period. Although the longest distances between consecutive 12-hr locations increased during the dark period, they continued increasing into early March when the sun was again visible, so this increase was probably unrelated directly to the light regimen. Conceivably the increased longer movements were related to the approaching breeding season which occurs in late March and early April Our pack's longest 12-hr, straight-line movement of 75.9 km may represent a record for a well-documented distance. There is one undocumented claim of wolves traveling 200 km in a day when hunted Our findings extend our knowledge of wolf routine travel capabilities considerably and help explain how wolves can survive as far north as they do, as well as why wolves were originally the most widely distributed non-human terrestrial mammal Our data also answer the question as to how wolves contend with the dark period in the highest latitudes of the species' range. We conclude that at least with the gross movements measurable by our methods, the 4-month period without direct sunlight produced little change in wolves' movement patterns.


FZD9 and MYOD1). In addition, we identified significant associations between three CpG sites (∼1%) and sex, including DNA methylation in CASP6, a gene that may respond to estradiol treatment, and in HSD17B12, which encodes a sex steroid hormone. Although we did not identify any significant associations between DNA methylation and the obesity measures, several nominally significant results were observed in genes related to adipogenesis, obesity, energy homeostasis and glucose homeostasis . In conclusion, we were able to replicate several findings from previous studies in our Mexican American cohort, supporting an important role for genetic effects on DNA methylation. In addition, we found a significant influence of age and sex on DNA methylation, and report on trend-level, novel associations between DNA methylation and measures of obesity.Several studies have identified effects of genetic variation on DNA methylation patterns and associated heritability, with research primarily focused on Caucasian individuals. In this paper, we examine the evidence for genetic effects on DNA methylation in a Mexican American cohort, a population burdened by a high prevalence of obesity. Using an Illumina-based platform and following stringent quality control procedures, we assessed a total of 395 CpG sites in peripheral blood samples obtained from 183 Mexican American individuals for evidence of heritability, proximal genetic regulation and association with age, sex and obesity measures (i.e. waist circumference and body mass index). We identified 16 CpG sites (∼4%) that were significantly heritable after Bonferroni correction for multiple testing and 27 CpG sites (∼6.9%) that showed evidence of genetic effects. Six CpG sites (∼2%) were associated with age, primarily exhibiting positive relationships, including CpG sites in two genes that have been implicated in previous genome-wide methylation studies of age ( DNMT1, DNMT3a and DNMT3b, regulate DNA methylation, which involves the covalent addition of a methyl group at the C(5) position of cytosine in cytosine-guanine (CpG) dinucleotides DNA methylation is an epigenetic modification that effectively regulates gene expression via gene silencing, and is thus considered an important mechanism in growth and development. Within humans, the DNA methyltransferases, Both genetic and epigenetic mechanisms can underlie disease development through gene dysregulation, and in many cases act synergistically. DNA methylation changes, at least at the single gene level, have been implicated in most complex diseases, including cancer 2); and waist circumference, which, as an index for central adiposity, has been shown to be a more accurate indicator of obesity, as well as an independent risk factor for diabetes Epigenetic regulation has not been well studied in Mexican Americans, who show a distinct disease profile relative to Caucasian populations. In particular, the prevalence of obesity, diabetes and metabolic syndrome are all elevated among Mexican Americans Written consent was obtained for all individuals in this study. Approval for this study and for the consent process was received from the Institutional Review Board at The University of Texas Health Science Center at San Antonio.st degree relatives, 221 pairs of 2nd degree relatives, 401 pairs of 3rd degree relatives, 320 pairs of 4th degree relatives and 39 pairs of 5th degree relatives. The mean age of this population is 42.15 years (standard deviation (sd): 13.74; range 19–75 years) and 55% of individuals are female. Each individual has been assessed for basic anthropometric measures, including those relevant to obesity, waist circumference (n = 182) and BMI (n = 183). In this study, the prevalence of obesity (defined as BMI≥30 kg/m2) is 59%, with 86% of the sample falling in the “overweight” category (BMI≥25 kg/m2). The average BMI for the cohort is 32.40 kg/m2 (sd: 7.40 kg/m2), and is 31.21 kg/m2 (sd: 6.81 kg/m2) for males and 33.38 kg/m2 (sd: 7.75 kg/m2) for females. The average waist circumference for the cohort is 104.86 cm (sd: 16.14 cm), and is 104.08 cm (sd: 14.91 cm) for males and 105.50 cm (sd: 17.12 cm) for females.The San Antonio Family Heart Study (SAFHS) was initiated in 1991 to study the effects of cardiovascular related illness in Mexican Americans GENE_Position_Strand, where GENE designates the gene name, Position designates the position of the CpG site relative to the transcription start site either within the promoter (P) or exon (E), and Strand designates whether the CpG site is on the forward (F) or reverse (R) strand.Using the EZ-96 DNA Methylation™ Kit  and following the manufacturers’ instructions, we performed bisulfite conversion of 1 µg DNA obtained from peripheral blood of an initially selected 188 Mexican American individuals following an overnight fast. We used Illumina GoldenGate technology incorporating the Methylation Cancer Panel I  to investigate the quantitative level of methylation at 1,505 CpG sites across 807 genes; 28.6% of genes contained only one CpG site, 57.3% contained two CpG sites and 14.1% of genes had three or more CpG sites. Although the CpG sites within this panel were chosen on the basis of their association or potential association with cancer, they fell within genes involved in DNA repair, cell cycle control, differentiation and apoptosis making them suitable for study in disorders other than cancer. The GoldenGate assay was carried out according to manufacturers’ instructions. Throughout the manuscript, CpG sites are annotated as th percentile, leaving a total of 183 samples for statistical analyses. Samples used in this study were also genotyped for approximately one million SNPs (see below) as part of a larger study; sex and familial relationships were verified during the genotyping process. Any CpG sites with a call rate less than 0.97 were excluded from analysis (n = 103). The platform used in this study has been shown to discriminate DNA methylation levels  that differ as little as 17% Initial analysis was carried out using GenomeStudio, applying background normalization by using the signals from built-in controls to minimize the amount of variation in background signals between arrays. DNA methylation for each CpG site is reported as a beta value, which is a score between 0 (completely unmethylated) and 1 (fully methylated). Built-in sample dependent and independent controls were assessed for each sample to look for any outliers. The raw data generated from GenomeStudio is given in −4 . The Infinium Whole-Genome Genotyping Assay was employed according to manufacturers’ instructions. Genotyping of the full SAFHS cohort included >2,000 samples, which underwent stringent quality control measures, excluding any samples with call rates <90% or that showed discrepancies in either sex or known familial relationships. According to these criteria, all 188 samples that underwent methylation profiling were eligible for genotyping analysis. Data cleaning in the full SAFHS population excluded monomorphic SNPs and those with a call rate less than 95%, those that were monomorphic, those whose minor allele was present in <10 individuals and those with Hardy-Weinberg Equilibrium test statistics of p≤10rd, 4th, 5th and 6th degree relatives are 0.0078, 0.0020, 0.0005 and 0.0001, respectively. The covariates sex, age, age2 and their interactions were used in all relevant analyses. Regression terms were estimated for each covariate, the statistical significance of which was assessed using a likelihood ratio test by comparing the log-likelihoods of models in which the covariate effect is estimated to the likelihoods of models in which the covariate effects were constrained to zero. Given that methylation profiling was performed on individuals that were chosen to maximize capture of founder genomes from the largest pedigrees and were optimally chosen for heritability inference, we calculated power to detect additive genetic heritability. We estimated that we have 50% power to detect heritability as small as 0.23 and 80% power to detect heritability as low as 0.36. To detect associations whilst correcting for multiple tests (Bonferroni correction) our power is 80% to detect variants that account for 16.7% of the variation. As such, we present results based on both Bonferroni correction and the less stringent false discovery rate (FDR) of 5%. The tail area-based estimation of FDR was computed using the R program “fdrtool” Our general computer package for statistical genetic analysis, SOLAR in silico conversions – reference, reverse complement, fully methylated and fully unmethylated. We then aligned the three probe sequences against these four reference genomes.We used the BSMAP software package PECAM1_E32_R and PECAM1_P135_F) could not be annotated and were excluded from this analysis. To test for genetic effects on DNA methylation levels, we took the measured-genotype approach to analyses and defined proximal SNPs as those within 100 kb of the flanking region around the genomic location of the CpG site . According to these criteria, all 393 CpG sites were assessed for genetic effects. For determining the statistical significance of a correlated SNP, the nominal p-value was estimated by comparing the log-likelihoods of a model with and without the SNP. Since we anticipated that many of the SNPs included in these analyses are likely to be in linkage disequilibrium, we used the method suggested by Li and Ji We employed the “measured genotype” model We have conducted a pilot study to examine the effects of genetic variation on DNA methylation in Mexican Americans, in which these effects, to our knowledge, have not yet been examined. Further, we assessed associations between DNA methylation and age and sex, as well as obesity measures, in order to better understand epigenetic contributions to health.To ensure DNA methylation levels were not significantly influenced by variation within the probe sequence, we tested for the presence of 39,706,715 SNPs derived from the 1000 Genomes Project. We identified SNPs in 163 of the 395 probes used for analysis see ; five pr−4; LTA_E28) was within a probe sequence that contained a SNP with a MAF of 35%, 19 bp from the CpG site  exhibited nominally significant (p<0.05) heritability, 28 (∼7%) were heritable at an FDR of 5% and 16 (∼4%) exhibited statistically significant heritability after Bonferroni correction for multiple testing  and may therefore be underpowered to accurately identify heritable factors influencing DNA methylation or may be prone to the detection of false positive results. A recent study by Breton, et al, AXL_P223 locus, suggesting that DNA methylation was not heritable but was highly influenced by environmental factors, however this too was performed in a small sample set of only 36 twins and as such is likely limited in its power to detect heritability. Nonetheless, given the overlap for several CpG sites across our study and that by Boks, et al, the case for DNA methylation being a heritable trait is becoming stronger and the application of heritability analysis to larger cohorts will ultimately be highly informative.Given the overlap in the platform and quality control strategies employed, we compared our heritability findings to those of Boks and colleagues, who examined heritability in a twin dataset using 430 CpG probes −5, 6.9%; ABL2_P459, GDF10_P95, HHIP_P307, LTA_E28; see LTA_E28 probe (rs2239704) was directly tested for association with DNA methylation levels and found to be significantly associated , applying a Sidak correction for 4,209 independent tests. In total, 214 statistically significant correlations were detected across 27 CpG sites (p<1.19×10−5), we found that DNA methylation levels within 6.9% of CpG sites tested were correlated with one or more SNPs. Although this is well in the range of the studies reported above, it is slightly higher than what was reported in the study by Boks, et al, (4%) using the same DNA methylation platform Recent genome-wide analyses have indicated that DNA methylation may indeed be tightly regulated by genetic factors. In fact, across studies 4–9% of CpG sites show evidence of genetic effects by SNPs within 50 kb-1 Mb of the site ALOX12_E85, ALOX12_P223, GNMT_P197_F, HPN_P823, MET_E333, NPR2_P618_F and SLC22A3_P634_F). In all cases where the same SNP was used to test for regulatory effects, the direction of effect was identical in both studies.Once again, we compared our regulatory findings to those presented by Boks, et al, who used the same platform but in a singleton dataset, examining 512 probes −4) showed at least nominal evidence for genetic effects; 10 of the 16 heritable CpG sites (63%) showed strong evidence for regulation by genetic variation. For nominally heritable CpG sites , 21% showed significant evidence for genetic effects and a further 65% showed nominal evidence for genetic effects; only 14% of nominally heritable sites showed no evidence of genetic effects  of the six associations that we identified was also identified as significant by Boks and colleagues We also tested for effects of age and sex on DNA methylation profiles and found substantial evidence for both; CARD15_P302 site showed a negative direction of effect (i.e. decreased methylation was associated with increased age), and this was the same direction of effect that was observed in the study by Boks, et al FZD9 and MYOD1) MYOD1 was associated with age in all three previously published studies, as well as in our own. Although two studies were performed in peripheral blood cells, the third assessed DNA methylation that was consistently associated with aging across four brain regions, suggesting that the DNA methylation changes associated with the MYOD1 gene may be general rather than tissue-specific. In addition to replicating results obtained from these genome-wide studies, we have identified new loci, possibly specific to the Mexican American population, whose DNA methylation status may be related to age. Due to the small number of samples assessed in our study however, such findings would need to be verified.Recent genome-wide studies have identified a number of age related differentially methylated regions with 78–98% of associated CpG sites showing positive associations CASP6_P201, showing increased methylation in women. Although a sex-specific role has not yet been defined, an early study in rats suggests that caspase-6 is involved in the control of apoptotic processes at estrus following treatment with the sex-hormone 17β-estradiol HSD17B12_P97, which encodes a sex steroid hormone, was also seen in females. HSD17B12 has been shown to be involved in the catalytic conversion of oestrone to estradiol and has also been implicated in fatty acid synthesis  could be mapped to the X-chromosome, using the reference, reverse complement, bisulfite converted fully methylated and bisulfite converted fully unmethylated genomes. Further, given some of the plausible biological roles discussed above, we believe that this study provides additional evidence for a potential role of DNA methylation in sex.Despite evidence for sexual dimorphism in a number of diseases, a recent genome-wide association study meta-analysis −4). In total, we identified 27 and 12 nominally significant (p<0.05) associations between DNA methylation and waist circumference and BMI, respectively  and BMI . MEST is well known to be involved in obesity and glucose metabolism. Gene expression increases have been shown in white adipose tissue of mice with diet-induced and genetically caused obesity/diabetes MEST has been shown in morbidly obese individuals as well as in newborns of mothers who had gestational diabetes NPY and waist circumference , but not with BMI. NPY is a hypothalamic peptide which regulates feeding behavior and energy homeostasis and has been proposed as a target for anti-obesity drugs NPY expression in the hypothalamus increases energy expenditure HOXA5 and BMI , but not waist circumference. HOXA5 has been implicated in body fat distribution, where it is more highly expressed in visceral  adipose tissue as opposed to subcutaneous  adipose tissue HOXA5 is up-regulated in abdominal subcutaneous adipose tissue following fat loss (one year after bariatric surgery) ARHGAP9CDKN2A (p16) FRZBJAK3PEG3CDKN2ASMARCB1Methylation levels of a CpG site within It is important to note the limitations of this study. As both the number of samples (n = 183) and number of CpG sites assessed (n = 395) are small, the power and scope of this study is limited. We have only assayed DNA methylation in one tissue type, peripheral blood, which may limit any generalized conclusions and may not be representative of changes that would be seen in diseased tissue (e.g. adipose tissue). However, given that lipid profiling, glucose measures and cardiovascular-related biomarkers are measured in blood, and that each of these are impacted by obesity, blood represents a good surrogate tissue for these analyses. Many of the results reported here further validate previously reported data and may also offer insight into any ethnic-specific differences.We examined the effects of genetic variation, sex and age on DNA methylation and assessed the role of DNA methylation in measures of obesity. Other studies have previously investigated the effects of genetic variation, age and sex using an identical or higher-throughput Illumina platform. However, this is the first study that has examined such factors in a Mexican American population. We have demonstrated here, further evidence supporting a role for genetic effects on DNA methylation, by replicating previous findings and presenting new data. Although several of our findings overlap with previous studies, the identification of novel CpG loci that are heritable, regulated by genetic variation, or that are influenced by age and sex may suggest the existence of ethnic-specific factors that drive regulation of DNA methylation. Such novel findings could also suggest that currently published studies, as well as our own, may be underpowered in their ability to detect true associations or have generated false positive results due to small sample sizes. Differences that are seen across studies could also be due to environmental effects, some of which may themselves be ethnic- or population-specific . We also provide nominal evidence that DNA methylation may be implicated in obesity in Mexican Americans, however additional follow up studies, particularly in a larger population are required to fully assess such implications.Table S1Raw data for 188 samples and 1,505 CpG sites.(ZIP)Click here for additional data file.Table S2List of all SNPs within probe sequences.(XLSX)Click here for additional data file.Table S3Heritability estimates for 395 CpG sites assessed.(XLSX)Click here for additional data file.Table S4Complete association analysis between all proximal genetic variants (defined as those within 100 kb) and 393 CpG sites.(XLSX)Click here for additional data file.Table S5Complete association analysis between age and DNA methylation at 395 CpG sites.(XLSX)Click here for additional data file.Table S6Complete association analysis between sex and DNA methylation at 395 CpG sites.(XLSX)Click here for additional data file.Table S7Complete association analysis between obesity measures (waist circumference and BMI) and DNA methylation at 395 CpG sites.(XLSX)Click here for additional data file.