Single‐nucleotide polymorphism discovery and validation in high‐density SNP array for genetic analysis in European white oaks

An Illumina Infinium SNP genotyping array was constructed for European white oaks. Six individuals of Quercus petraea and Q. robur were considered for SNP discovery using both previously obtained Sanger sequences across 676 gene regions (1371 in vitro SNPs) and Roche 454 technology sequences from 5112 contigs (6542 putative in silico SNPs). The 7913 SNPs were genotyped across the six parental individuals, full‐sib progenies (one within each species and two interspecific crosses between Q. petraea and Q. robur) and three natural populations from south‐western France that included two additional interfertile white oak species (Q. pubescens and Q. pyrenaica). The genotyping success rate in mapping populations was 80.4% overall and 72.4% for polymorphic SNPs. In natural populations, these figures were lower (54.8% and 51.9%, respectively). Illumina genotype clusters with compression (shift of clusters on the normalized x‐axis) were detected in ~25% of the successfully genotyped SNPs and may be due to the presence of paralogues. Compressed clusters were significantly more frequent for SNPs showing a priori incorrect Illumina genotypes, suggesting that they should be considered with caution or discarded. Altogether, these results show a high experimental error rate for the Infinium array (between 15% and 20% of SNPs potentially unreliable and 10% when excluding all compressed clusters), and recommendations are proposed when applying this type of high‐throughput technique. Finally, results on diversity levels and shared polymorphisms across targeted white oaks and more distant species of the Quercus genus are discussed, and perspectives for future comparative studies are proposed.     

Lack of Evidence for the Role of Human Adenovirus‐36 in Obesity in a European Cohort

Adenovirus infection has been shown to increase adiposity in chickens, mice, and nonhuman primates. Adenovirus type 36 (Ad‐36) DNA was detected in adipose tissues in these animal trials. In the United States, Ad‐36 significantly correlates with obesity as illustrated by an Ad‐36 seroprevalence of 30% in obese individuals and 11% in nonobese individuals. We investigated the possibility of a similar correlation of Ad‐36 in Dutch and Belgian persons. In total, 509 serum samples were analyzed for Ad‐36 antibodies using a serum neutralization assay. In addition, PCR was used to detect adenoviral DNA in visceral adipose tissue of 31 severely obese surgical patients. Our results indicated an overall Ad‐36 seroprevalence of 5.5% increasing with age. BMI of Ad‐36 seropositive humans was not significantly different from seronegative humans. No adenoviral DNA could be found using PCR on visceral adipose tissue. In conclusion, this first Ad‐36 study in the Netherlands and in Belgium indicates that Ad‐36 does not play a role as a direct cause of BMI increase and obesity in humans in Western Europe.     

Single‐nucleotide and long‐patch base excision repair of DNA damage in plants

Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through in vitro reproduction of the entire repair reaction in cell‐free extracts. However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5′‐ or 3′‐blocked ends, respectively. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short‐patch BER) or several nucleotides (long‐patch BER). This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways.     

Single‐nucleotide polymorphism discovery and panel characterization in the African forest elephant

The continuing decline in forest elephant (Loxodonta cyclotis) numbers due to poaching and habitat reduction is driving the search for new tools to inform management and conservation. For dense rainforest species, basic ecological data on populations and threats can be challenging and expensive to collect, impeding conservation action in the field. As such, genetic monitoring is being increasingly implemented to complement or replace more burdensome field techniques. Single‐nucleotide polymorphisms (SNPs) are particularly cost‐effective and informative markers that can be used for a range of practical applications, including population census, assessment of human impact on social and genetic structure, and investigation of the illegal wildlife trade. SNP resources for elephants are scarce, but next‐generation sequencing provides the opportunity for rapid, inexpensive generation of SNP markers in nonmodel species. Here, we sourced forest elephant DNA from 23 samples collected from 10 locations within Gabon, Central Africa, and applied double‐digest restriction‐site‐associated DNA (ddRAD) sequencing to discover 31,851 tags containing SNPs that were reduced to a set of 1,365 high‐quality candidate SNP markers. A subset of 115 candidate SNPs was then selected for assay design and validation using 56 additional samples. Genotyping resulted in a high conversion rate (93%) and a low per allele error rate (0.07%). This study provides the first panel of 107 validated SNP markers for forest elephants. This resource presents great potential for new genetic tools to produce reliable data and underpin a step‐change in conservation policies for this elusive species.     

Single‐nucleotide polymorphism discovery and diversity in the model legume Medicago truncatula

Extensive genomic resources are available in the model legume Medicago truncatula. Here, we present the discovery and design of the first array of single‐nucleotide polymorphism (SNP) markers in M. truncatula through large‐scale Sanger resequencing of genomic fragments spanning the genome, in a diverse panel of 16 M. truncatula accessions. Both anonymous fragments and fragments targeting candidate genes for flowering phenology and symbiosis were surveyed for nucleotide variation in almost 230 kb of unique genomic regions. A set of 384 SNP markers was designed for an Illumina's GoldenGate assay, genotyped on a collection of 192 inbred lines (CC192) representing the geographical range of the species and used to survey the diversity of two natural populations. Finally, 86% of the tested SNPs were of high quality and exhibited polymorphism in the CC192 collection. Even at the population level, we detected polymorphism for more than 50% of the selected SNPs. Analysis of the allele frequency spectrum in the CC192 showed a reduced ascertainment bias, mostly limited to very rare alleles (frequency <0.01). The substantial polymorphism detected at the species and population levels, the high marker quality and the potential to survey large samples of individuals make this set of SNP markers a valuable tool to improve our understanding of the effect of demographic and selective factors that shape the natural genetic diversity within the selfing species Medicago truncatula.     

Treatment of Morbid Obesity in Low‐income Adolescents: Effects of Parental Self‐monitoring

Objective: This study examined the extent to which consistency of self‐monitoring by participants and their parents was related to weight control over an initial period of 3 months within the context of a treatment program for morbidly obese low‐income minority adolescents. Research Methods and Procedures: Eighty‐three obese adolescents (mean age, 13.0 years; 51% boys; 92% African American; mean BMI, 43.0 kg/m²; mean BMI z‐score, 6.0) and at least one parent participated in a long‐term treatment program that included a very‐low‐fat dietary focus, weekly group cognitive‐behavior therapy, monthly nutrition education classes, a 12‐week physical therapy class, and medical monitoring. Results: Participants who self‐monitored on the majority of days compared with those who did not self‐monitor at all or who self‐monitored infrequently attended more sessions and generally lost more weight over the first 3 months. Although parents signed behavioral contracts committing to self‐monitor their own eating and exercising over the first month, only 12% did so. Nonetheless, participants whose parents self‐monitored were much more likely to self‐monitor consistently and lose weight during the first 3 months. Discussion: These results indicate that self‐monitoring is a cornerstone of successful weight control even for morbidly obese low‐income minority adolescents; targeting consistency of self‐monitoring among these high‐risk weight controllers and their parents should be just as important as it is for more affluent and less overweight adolescents.     

Two‐Dimensional Strain and Twist by Vector Velocity Imaging in Adolescents With Severe Obesity

The prevalence of severe obesity is increasing worldwide in adolescents. Whether it is associated with functional myocardial abnormalities remains largely unknown, potentially because of its frequent association with other cardiovascular risk factors and also use of insensitive techniques to detect subclinical changes in myocardial function. We used 2D vector velocity imaging (VVI) to investigate early changes in left ventricular (LV) myocardial function in youths with isolated severe obesity. Thirty‐seven asymptomatic severely obese adolescents free of diabetes and hypertension, and 24 lean controls were enrolled. LV longitudinal, basal, and apical circumferential strain, strain rate (SR), rotations, and LV twist were measured. Obese adolescents had greater LV mass and reduced systolic and early diastolic tissue Doppler imaging (TDI) velocities than lean counterparts. L strain (?24%) and systolic and early diastolic SR were also diminished in the obese, whereas no intergroup differences existed for the circumferential deformation indexes. LV twist was more pronounced in the obese (+1.7°, P < 0.01) on account of greater apical rotation only (4.1 ± 0.9 vs. 5.2 ± 1.2°, P < 0.01), potentially compensating for the loss in longitudinal function. Systolic—diastolic coupling, an important component of early filling and diastolic function, was maintained with severe obesity. No intergroup differences were reported regarding time to peak values for all VVI indexes highlighting that dynamics of strain and twist/untwist along the cardiac cycle was preserved with severe obesity. Isolated severe obesity in adolescents, at a preclinical stage, is associated with changes in myocardial deformation and torsional mechanics that could be in part related to alterations in relaxation and contractility properties of subendocardial fibers.     

Investigation of the Locus Near MC4R With Childhood Obesity in Americans of European and African Ancestry

Recently a modest, but consistently, replicated association was demonstrated between obesity and the single‐nucleotide polymorphism (SNP), rs17782313, 3′ of the MC4R locus as a consequence of a meta‐analysis of genome‐wide association (GWA) studies of the disease in white populations. We investigated the association in the context of the childhood form of the disease utilizing data from our ongoing GWA study in a cohort of 728 European‐American (EA) obese children (BMI ≥95th percentile) and 3,960 EA controls (BMI <95th percentile), as well as 1,008 African‐American (AA) obese children and 2,715 AA controls. rs571312, rs10871777, and rs476828 (perfect surrogates for rs17782313) yielded odds ratios in the EA cohort of 1.142 (P = 0.045), 1.137 (P = 0.054), and 1.145 (P = 0.042); however, there was no significant association with these SNPs in the AA cohort. When investigating all 30 SNPs present on the Illumina BeadChip at this locus, again there was no evidence for association in AA cases when correcting for the number of tests employed. As such, variants 3′ to the MC4R locus present on the genotyping platform utilized confer a similar magnitude of risk of obesity in white children as to their adult white counterparts but this observation did not extend to AAs.     

Single‐nucleotide polymorphism discovery in Leptographium longiclavatum,a mountain pine beetle‐associated symbiotic fungus,using whole‐genome resequencing

Single‐nucleotide polymorphisms (SNPs) are rapidly becoming the standard markers in population genomics studies; however, their use in nonmodel organisms is limited due to the lack of cost‐effective approaches to uncover genome‐wide variation, and the large number of individuals needed in the screening process to reduce ascertainment bias. To discover SNPs for population genomics studies in the fungal symbionts of the mountain pine beetle (MPB), we developed a road map to discover SNPs and to produce a genotyping platform. We undertook a whole‐genome sequencing approach of Leptographium longiclavatum in combination with available genomics resources of another MPB symbiont, Grosmannia clavigera. We sequenced 71 individuals pooled into four groups using the Illumina sequencing technology. We generated between 27 and 30 million reads of 75 bp that resulted in a total of 1, 181 contigs longer than 2 kb and an assembled genome size of 28.9 Mb (N₅₀ = 48 kb, average depth = 125x). A total of 9052 proteins were annotated, and between 9531 and 17 266 SNPs were identified in the four pools. A subset of 206 genes (containing 574 SNPs, 11% false positives) was used to develop a genotyping platform for this species. Using this roadmap, we developed a genotyping assay with a total of 147 SNPs located in 121 genes using the Illumina^® Sequenom iPLEX Gold. Our preliminary genotyping (success rate = 85%) of 304 individuals from 36 populations supports the utility of this approach for population genomics studies in other MPB fungal symbionts and other fungal nonmodel species.     

Physiological and pathological roles of a multi-ligand receptor CD36 in atherogenesis; insights from CD36-deficient patients

Oxidized low density lipoprotein (LDL) (Ox-LDL) plays an important role in the pathogenesis of atherosclerosis. Oxidized LDL is taken up by macrophages via scavenger receptors. CD36 is an 88 kDa glycoprotein expressed on platelets, monocyte-macrophages, microvascular endothelial cells, adipose tissue, skeletal muscles and heart. We found patients with CD36 deficiency and identified several mutations in the CD36 gene. We also reported that CD36-deficient macrophages showed a 50% reduction in the binding of Ox-LDL, suggesting that CD36 is one of the major receptors for Ox-LDL. CD36 was expressed on macrophages in the atherosclerotic lesions of human aorta and coronary arteries especially on foamed macrophages. The distribution of CD36 expression was slightly different from that of scavenger receptor class A types I and II. The expression of CD36 on macrophages was up-regulated by Ox-LDL and down-regulated by interferon gamma. Since CD36 is a transporter of long-chain fatty acids (LCFA), CD36-deficient patients showed a defect in the uptake of an LCFA analog, BMIPP, by the heart. Furthermore, the secretion of IL-1beta and TNF-alpha from monocyte-derived macrophages induced by Ox-LDL was markedly reduced and the activation of NF-kappaB was attenuated in CD36-deficient subjects compared with controls, suggesting that CD36-mediated signaling is also impaired in CD36 deficiency.To elucidate the roles of CD36 in vivo, we characterized the clinical profile of CD36-deficient patients. Most of them were accompanied by hyperlipidemia (mainly hypertriglyceridemia), increased remnant lipoproteins and mild elevation of fasting plasma glucose level and blood pressure. Glucose clamp technique revealed mean whole body glucose uptake was reduced in CD36-deficient patients, indicating the presence of insulin resistance. The frequency of CD36 deficiency was higher in patients with coronary heart disease (CHD) than in control subjects. Taken together, CD36 deficiency is accompanied by (1) hyperlipidemia and increased remnant lipoproteins, (2) impaired glucose metabolism based upon insulin resistance, and (3) mild hypertension, and comprises one of the genetic backgrounds of the metabolic syndrome, leading to the development of CHD.     

5‐HT2A Receptor Gene Promoter Polymorphism in Relation to Abdominal Obesity and Cortisol

Objective: There is considerable evidence that cortisol secretion is associated with obesity. The regulation of the 5‐hydroxytryptamine receptor 2A (5‐HT_2A) gene might play an essential role because it is involved in the control of cortisol secretion. Therefore, we examined the potential impact of the 5‐HT_2A ?1438G/A promoter polymorphism on obesity and estimates of insulin, glucose, and lipid metabolism as well as circulating hormones, including salivary cortisol, in 284 unrelated Swedish men born in 1944. Research Methods and Procedures: The subjects were genotyped by using polymerase chain reaction amplification of the promoter region of the gene for 5‐HT_2A followed by digestion of the reaction product with the restriction enzyme MspI. Results: The frequencies were 0.39 for allele ?1438A and 0.61 for allele ?1438G. Homozygotes for the ?1438G allele had, in comparison with ?1438A/A subjects, higher body mass index, waist‐to‐hip ratio, and abdominal sagittal diameter. Moreover, cortisol escape from 0.25‐mg dexamethasone suppression was found in subjects with the ?1438A/G genotype. Serum leptin, fasting insulin, and glucose, as well as serum lipids, were not different across the ?1438G/A genotype groups. Discussion: From these results, we suggest the possibility that an abnormal production rate of the 5‐HT_2A gene product might lead to the development of abdominal obesity. The pathophysiology could involve stress factors that destabilize the serotonin‐hypothalamic‐pituitary‐adrenal system in those with genetic vulnerability in the serotonin receptor gene.     

Association of the European Lactase Persistence Variant (LCT-13910 C>T Polymorphism) with Obesity in the Canary Islands

Background

European lactose tolerance genotype (LCT -13910 C>T, rs4988234) has been positively associated to body mass indexes (BMI) in a meta-analysis of 31,720 individuals of northern and central European descent. A strong association of lactase persistence (LP) with BMI and obesity has also been traced in a Spanish Mediterranean population. The aim of this study was to analyze a potential association of LP compared to lactase non-persistence (LNP) with BMI in inhabitants of the Canary Islands of Spain using Mendelian randomization.

Methods

A representative, randomly sampled population of adults belonging to the Canary Islands Nutrition Survey (ENCA) in Spain, aged 18–75 years (n = 551), was genotyped for the LCT – 13910 C>T polymorphism. Milk consumption was assessed by a validated questionnaire. Anthropometric variables were directly measured. WHO classification of BMI was used.

Results

LP individuals were significantly more obese than LNP subjects (χ² = 10.59; p<0.005). LP showed in a multivariate linear regression analysis showed a positive association of LP with BMI compared to LNP, (β = 0.96; 95% CI: 0.08–1.85, p = 0.033). In a multinomial logistic regression analysis normal range weight LP subjects showed an odds ratio for obesity of 2.41; 95%CI 1.39–418, (p = 0.002) compared to LNP.

Conclusions

The T-13910 of the allele LCT-13910 C>T polymorphism is positively associated with BMI. LP increases significantly the risk to develop obesity in the studied population. The LCT-13910 C>T polymorphism stands proxy for the lifetime exposure pattern, milk intake, that may increase susceptibility to obesity and to obesity related pathologies.     

SR‐BI,CD36, and caveolin‐1 contribute positively to cholesterol efflux in hepatic cells

In non‐hepatic cells, scavenger receptor class B type I (SR‐BI), cluster of differentiation 36 (CD36), and caveolin‐1 were described as mediators of cholesterol efflux, the first step of reverse cholesterol transport (RCT). Stable transformants of HepG2 cells overexpressing SR‐BI, CD36, or caveolin‐1 were generated, as well as cells overexpressing both caveolin‐1 and SR‐BI or caveolin‐1 and CD36 in order to address the effect of caveolin‐1 on both receptor activities. These cells were analyzed for their ability to efflux cholesterol to HDL₃. Our results show that overexpressing SR‐BI, CD36, or caveolin‐1 increases cholesterol efflux by 106, 92, and 48%, respectively. Moreover, the dual overexpressions of caveolin‐1 and SR‐BI or caveolin‐1 and CD36 lead to a more prominent increase in cholesterol efflux. Studies were also conducted with primary cultures of SR‐BI knockout (KO), CD36 KO, and SR‐BI/CD36 double‐KO (dKO) mice. SR‐BI KO and SR‐BI/CD36 dKO hepatic cells show 41 and 56% less cholesterol efflux, respectively, than normal hepatic cells. No significant difference was observed between the efflux of normal and CD36 KO cells. The difference between the role of human and murine CD36 correlated with the absence of CD36 dimers in mouse caveolae/rafts. Overall, our results show that SR‐BI is clearly involved in cholesterol efflux in mouse and human hepatic cells, while CD36 plays a significant role in human cells. Copyright © 2010 John Wiley & Sons, Ltd.