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Heritability of obesity is substantial and recent meta-analyses of genome-wide association studies (GWASs) have been successful in detecting several robustly associated genomic regions for obesity using single-nucleotide polymorphisms (SNPs). However, taken together, the SNPs explain only a small proportion of the overall heritability. Copy number variations (CNVs) might contribute to the 'missing heritability'. We searched genome-wide for association between common CNVs and early-onset extreme obesity. Four hundred and twenty-four case-parents obesity trios and an independent sample of 453 extremely obese children and adolescents and 435 normal-weight and lean adult controls were genotyped by the Affymetrix Genome-Wide Human SNP Array 6.0. We detected 20 common copy number variable regions (CNVRs) which were associated with obesity. The most promising CNVRs were followed-up in an independent sample of 365 obesity trios, confirming the association for two candidate CNVRs. We identified a common CNVR exclusively covering the three olfactory receptor genes OR4P4, OR4S2 and OR4C6 to be associated with obesity (combined P-value = 0.015 in a total of 789 families; odds ratio for the obesity effect allele = 1.19; 95% confidence interval = 1.016–1.394). We also replicated two common deletions (near NEGR1 and at chromosome 10q11.22) that have previously been reported to be associated with body weight. Additionally, we support a rare CNV on chromosome 16 that has recently been reported by two independent groups. However, rare CNVs had not been the focus of our study. We conclude that common CNVs are unlikely to contribute substantially to the genetic basis of early-onset extreme obesity.
Obesity is a heritable complex trait (1,2). Genome-wide association studies (GWASs) led to the identification of various common single-nucleotide polymorphisms (SNPs) for human obesity (3–8). Although most of the results of GWASs have been highly reproducible, they explain only a minor fraction of the variance of body mass index (BMI) when compared with the total expected heritability of BMI [~50%, (9)]. Hence, a substantial 'missing heritability' (10) becomes obvious, which might in part be explained by copy number variants (CNVs). CNVs are by definition chromosomal regions with sizes of 1kb to several megabases (Mb) being interindividually present in variable numbers. At a genome-wide level, thousands of CNVs have already been identified. Owing to resolution of the current technology, most of these CNVs are >5 kb (11). The database of Genomic Variants (http://projects.tcag.ca/variation/) currently lists 57 829 CNVs at 14 478 CNV loci and half of these CNVs are of sizes from 1 to 10 kb. The frequency spectrum and the precise structure of CNVs are closely related to the technical and algorithmic methods applied (12,13). Specific CNVs have been related to a number of complex traits–examples are psoriasis (14), schizophrenia (15), autism (16), developmental disorders (17) or HIV1/AIDS susceptibility (18). In some instances, particular CNVs were related to certain disorders as de novo events [e.g. schizophrenia (15)].
Currently, two common CNV regions have been described for BMI and obesity. At first, association of a common deletion near the neuronal growth regulator 1 gene (NEGR1) with BMI was reported (8). Some CNVs are in strong linkage disequilibrium with adjacent SNPs; therefore, this CNV was detected in a meta-analysis of SNP data of 15 GWAS comprising more than 32 000 individuals. Recently, another common CNV (chromosome 10p11.22) was shown to be associated with BMI (19) in a Chinese sample (nominal P-value = 0.011).
In addition to these common CNVs, three recent reports (20–22) underlined the importance of rare, large CNVs for body weight regulation. Two of these studies (20,21) depicted a genomic region on chromosome 16p11.2 which harbours highly penetrant microdeletions (~500 kb) associated with (extreme) obesity. This region was previously reported to be associated with autism and mental retardation (20,21,23); in fact, some of the obese patients analysed by Bochukova et al. (20) additionally had developmental disorders. However, the association of these microdeletions and obesity was also found in individuals ascertained for obesity only (21), suggesting a possible direct association of the deletions at 16p11.2 with obesity apart from the cognitive phenotype. Wang et al. (22) performed a genome-wide CNV survey focusing on large (>1 Mb) CNVs, which were found to be over-represented in case versus control subjects. However, to explain a substantial part of the 'missing heritability', thousands of such rare CNVs have to be assumed. Recent considerations (24,25) showed that even meta-analyses of large-scale consortia will be underpowered to detect most of these multiple, rare variants.
The potential of common CNVs has until recently (11,26) been largely neglected despite the fact that power issues to detect common CNVs will be less extreme when compared with rare CNVs. The main reason might be that samples have often been genotyped by arrays not designed for the detection of common CNVs. As an example, following recent reports (13), 44% of the common CNVs detectable by the more recent Affymetrix Genome-Wide Human SNP Array 6.0 would not have been detected by a previous Affymetrix chip (Affymetrix 500K Mapping Array Set).
Here we focus on common CNVs and report a genome-wide CNV detection and association analysis for early-onset extreme obesity using two GWAS discovery data sets (family-based and case–control, altogether 2160 genotyped individuals) and an additional replication sample of 365 independent obesity trios (Fig. 1).
We observed 244 autosomal common CNVRs; out of which 240 were at least partially listed in the Database of Genomic Variants (http://projects.tcag.ca/variation). The four yet-unknown CNVRs are positioned on chromosomes 1q25.1 (at position 173 063 167–173 068 476 bp, hg18), 3p12.1 (84 782 436–84 784 839 bp; hg18), 4q27 (122 501 906–122 504 445 bp; hg18) and 14q11.1 (18 072 112–18 183 975 bp; hg18). With an average and median size of 183.92 and 13.97 kb, respectively, these 244 common CNVRs cover 1.56% (44.88 Mb) of the human genome (Table 1).
Among the 244 common CNVRs, we detected 25 regions that showed suggestive evidence for an association to (extreme) obesity in the family-based GWAS discovery sample (two-sided asymptotic P-value in the family-based association test for CNVs [CNV-FBAT] ≤ 0.05; Table 2 and Fig. 2). To assess the overall impact of the 25 CNVRs on obesity, we contrasted the effects of their correspondingly involved CNV markers to those of randomly sampled sets of CNV markers chosen from all CNV markers covering the analysed 244 common CNVRs. In a QQ plot (Supplementary Material, Fig. S1), we compared the distribution of the observed association P-values for the 1314 CNV markers covering the 25 detected CNVRs to the distribution of 1000 random sets of P-values for 1314 CNV markers each (expectation under the null hypothesis). We observed stronger obesity association signals for the 25 detected CNVRs when compared with the random distribution (Supplementary Material, Fig. S1).
These 25 CNVRs were followed up by investigating an independent case–control GWAS sample: 20 of the 25 CNVRs also indicated evidence for a directionally consistent association with obesity (one-sided P ≤ 0.025; 5 of the 20 CNVRs with a Bonferroni-corrected CNVR-adjusted one-sided P ≤ 0.025; Table 2). Performing 10 000 permutations of the case–control status, we observed only two permutation samples with 20 or more of such directionally consistent associations with obesity (empirical P-value 2 × 10−04). For 3 of the 20 CNVRs [CNVR 10q11.22 (1), CNVR 16p13.11 and CNVR 17q21.31], association signals for obesity were found at exactly the same CNV marker in both GWAS discovery samples. In the remaining 17 CNVRs, at least one CNV marker showed evidence for an association with obesity in the family-based GWAS sample, while at least one other correlated CNV marker in the case–control GWAS sample similarly showed evidence for an obesity association signal. The maximal pairwise correlation coefficient between each of these two CNV markers associated with obesity ranged from 0.24 to 0.91 as estimated in the parents, whereas the range of pairwise correlations between those two markers with minimal P-values in the family-based and in the case–control GWAS discovery sample was 0.15–0.84. Among the 20 CNVRs associated with obesity, reduced copy numbers (deletions) were associated with obesity in 18 CNVRs (Table 2).
For replication analysis of the signals in the 20 CNVRs with evidence for an association in both GWAS samples, we focused on a CNVR-tagging SNP approach (see Materials and Methods section). For two (CNVR 11q11 and CNVR 1p31.1) of these regions, a tagging SNP could be identified, which has been analysed in a third independent sample of 365 obesity trios. In addition, we also screened the 20 CNVRs in a subsample of the 365 obesity trios (281 obesity trios) using additional Affymetrix Genome-Wide Human SNP Array 6.0 data (array-based replication).
The CNVR on chromosome 11q11 (CNVR 11q11) was the best CNVR (according to P-value in the family-based GWAS sample; min. Pfamily-based = 0.0047) among the set of 20 CNVRs which were directionally consistently associated with obesity in both GWAS discovery samples (with correlation coefficients up to r = 0.75 for pairs of associated CNV markers in the two samples; Table 2). CNVR 11q11 was similarly supported in the multi-marker analysis (min. P = 8.66 × 10−4; Fig. 3). This CNVR represents a locus previously not reported to be associated with obesity. It is located between 55 130 596 and 55 210 165 bp spanning ~80 kb. Interestingly, the region exclusively covers three olfactory receptor (OLR) genes: OR4P4, OR4S2 and OR4C6 (according to RefSeq of UCSC hg18; http://genome.ucsc.edu/) all belonging to the OLR family 4.
We followed CNVR 11q11 up in the replication sample of 365 independent obesity trios due to the availability of a CNV-tagging SNP (rs9804659) and observed a directionally consistent trend for an association to obesity with a one-sided P-value of 0.076 [odds ratio for the obesity effect allele (OR) = 1.188; 95% confidence interval (CI) = 0.939–1.502]. The maximal squared Pearson correlation (r2) between rs9804659 and any of the CNV markers in CNVR 11q11 was 0.65, while r2 was 0.53 between rs9804659 and the best associated CNV marker (minimal P-value in the family-based discovery sample; Table 3). Thus CNVR 11q11 is not that well tagged by rs9804659, which might explain why the P-value in the replication is an order of magnitude lower than in the discovery sample.
Consequently, given this moderate correlation, for technical validation and further analyses, we re-assessed CNVR 11q11 by quantitative real-time polymerase chain reaction (qPCR) in the family-based GWAS discovery sample and in the family-based replication sample of 365 case-parents obesity trios. On the basis of the qPCR findings, we validated CNVR 11q11 as a biallelic deletion region with a minor allele frequency of 28% in the set of all 789 parental pairs. We observed 7.71% homozygotes and 40.35% heterozygotes for the deletion in the parents. In obese offspring, we detected 9.72% homozygotes and 40.03% heterozygotes. Thus, the number of homozygous deletions was slightly increased in the obese offspring, which was consistent with the observed transmission disequilibrium. The analysis of the qPCR-validated copy number calls in the family-based GWAS discovery sample indicated a trend towards preferable transmission of the 11q11 deletion to obese children (OR = 1.171; 95% CI = 0.947–1.448; one-sided P = 0.066). A similar qPCR-based analysis in the replication sample revealed a directionally consistent finding (OR = 1.214; 95% CI = 0.959–1.537; one-sided P = 0.056). Finally, we performed a joint analysis of all case-parents trios (discovery and replication sample) to increase the precision of the estimator, resulting in an OR of 1.190 for the deletion allele (95% CI = 1.016–1.394; one-sided P = 0.015).
All 20 CNVRs with evidence for an association with obesity in both GWAS discovery samples were followed up in 281 independent obesity trios (a subsample of the 365 independent obesity trios analysed at the two tagging SNPs) that were very recently genotyped (Affymetrix Genome-Wide Human SNP Array 6.0). Six of the 20 CNVRs again showed evidence for a directionally consistent association with obesity (one-sided CNV-FBAT P-value ≤ 0.025; Table 2). These six CNVRs involve those two CNVRs that are also supported by the tagging SNP approach. Furthermore, association signals for only these two CNVRs were found at exactly the same CNV markers as in the family-based discovery GWAS sample. Neither in the family-based discovery nor in the array-based replication sample did any of the CNVRs achieve significant CNVR-adjusted P-values (all >0.025; data not shown). However, in a combined analysis of both family-based samples, three CNVRs presented at least nominally significant CNVR-adjusted one-sided P-values (≤0.025; Table 2). None of these CNVR associations remained significant after strict Bonferroni correction, which may potentially be due to an insufficient sample size. Among the three CNVRs with nominally significant CNVR-adjusted P-values, again those two CNVRs tagged by SNPs were found. Additional support was given for CNVR 4q32.1, which does not comprise any gene.
CNVR 1p31.1 (Table 2) 20 kb upstream of NEGR1 was the only CNV associated with BMI in a meta-analysis of 15 GWAS (8). Identification of the CNV was based on SNP tagging in a large meta-analysis of population-based samples of European origin. We were able to confirm this CNVR in both of our GWAS discovery samples (min. Pfamily-based = 0.0084; min. Pcc = 0.0068 for different CNV markers) while we observed stronger signals in the family-based GWAS sample (Fig. 4). Moreover, CNVR 1p31.1 is one of the two CNVRs (out of 20) that could be tagged by an SNP (rs2815752). The major allele T of rs2815752 is tagging the deletion allele of CNVR 1p31.1. Analysis of the CNVR-tagging SNP in the replication sample (365 independent obesity trios) again underlined the obesity association (OR = 1.277; 95% CI = 1.021–1.597; one-sided P-value = 0.0157; Table 3). In a joint analysis of the two family-based samples at rs2815752, we estimated an OR of 1.214 (95% CI = 1.046–1.408, one-sided P-value = 0.0052). Additional support for this CNVR was provided by the array-based replication analysis (see above).
In addition to CNVR 1p31.1, the other previously reported common CNVR associated with BMI (nominal P-value 0.011) was located on chromosome 10q11.22 which was initially described in an adult Chinese population-based sample (19). In our study, this CNVR [CNVR 10q11.22 (1)] was also among those 20 candidates with evidence for association with obesity in both GWAS discovery samples. In addition, this CNVR is among those three candidates with evidence for association at exactly the same CNV marker (located between 46 478 786 to 46 478 811 bp with a combined P-value = 1.13 × 10−6 in the two GWAS samples). In the case–control GWAS sample, the number of associated CNV markers was relatively large (47 of 161 CNV markers with P ≤ 0.025; Fig. 5). Moreover, CNVR 10q11.22 (1) is among those 6 CNVRs with a minimal one-sided CNV-FBAT P-value ≤ 0.025 in the array-based replication analyses. CNVR 10q11.22 (1) (Table 2) covers four genes: (i) the pancreatic polypeptide receptor 1 (PPYR1) was a member of the seven transmembrane domain-G-protein-coupled receptor family and pancreatic polypeptide (PP) is a potent anti-obesity agent known to inhibit food intake (27), (ii) Synaptotagmin XV (SYT15), (iii) the G protein regulated inducer of neurite outgrowth 2 (GPRIN2) and (iv) the heterogeneous nuclear ribonucleoprotein A1 pseudogene (LOC728643).
Beyond the focus of this study on common CNVRs for early-onset (extreme) obesity, we also explored selected regions harbouring rare CNVs (20,21). First, we looked at deletions on chromosome 16p11.2 (28.4–31.0 Mb) of at least 593 kb in both of our GWAS samples (21). In the family-based GWAS sample, we exclusively found one obese patient with a heterozygous de novo deletion of 709 kb. Moreover, in the case–control GWAS sample, four obese cases but none of the controls were called for heterozygous deletions with sizes between 709 and 894 kb (one-sided P-value of Fisher's exact test = 0.13). Secondly, we compared the list of rare recurrent CNVs found in patients with severe early-onset obesity (20) to the common CNVRs determined here. We observed no exact overlap, which was not unexpected given our focus on common CNVs. However, 2 of the 12 CNVs reported for patients with severe early-onset extreme obesity (20) were 133 kb downstream and 66 kb upstream of the CNVRs detected here (CNVR 3p11.1 and CNVR 15q13.2; Table 2). Both these CNVRs showed evidence for association with obesity in both of our GWAS discovery samples (3p11.1: min. Pfamily-based = 0.019; min. Pcc = 0.0072 for different CNV markers with maximum correlation r = 0.68; 15q13.2: min. Pfamily-based = 0.013; min. Pcc = 5.5 × 10−7 for different CNV markers with maximum correlation r = 0.70).
We performed a genome-wide analysis of common copy number variations (CNVs) in two discovery samples ascertained for early-onset (extreme) obesity. We observed 244 CNVRs that covered ~1.56% of the genome-most of these regions were already known from databases. Among the 244 CNVRs, we detected 20 regions that were directionally consistently associated with obesity in both discovery samples.
Among these 20 associated regions, we identified a new CNVR 11q11 associated with early-onset (extreme) obesity. While the presence of copy number variability at CNVR 11q11 is well established (28–30), its relationship to obesity has not been described before. CNVR 11q11 covers the three protein coding genes OR4P4, OR4S2, OR4C6 (according to RefSeq of UCSC hg18; http://genome.ucsc.edu/) of the OLR family 4. OLRs interact with odorant molecules in the nose, to initiate a neuronal response that triggers the perception of a smell. They form the largest mammalian protein superfamily. There is a high percentage (~55%) of human pseudogenes (29). The OLR proteins are members of the family of G-protein-coupled receptors arising from single-coding exon genes.
It was hypothesized that CNVs play an important role in the evolution of the human olfactory repertoire (29). About half of the CNVs affecting the human OLR repertoire involve more than one OLR. It was also observed that CNVs were more frequent among pseudogenes of OLRs than among functional genes (29). Comparison to the chimpanzee, reference genome revealed that all of the detected deletion alleles are human derived. This indicated a profound effect of human-specific deletions on the individual OLR gene content. It was suggested that these deletion alleles may be used in future genetic association studies of olfactory inter-individual differences (29). Evidence was provided for OLR enrichment in CNVs not being due to positive selection but due to the dominance of OLR in segmentally duplicated regions (30). Additionally, purifying selection against CNVs is lower in regions containing OLRs than in regions containing essential genes (30). A possible link between OLRs and obesity has previously been suggested based on the observation of altered olfactory acuity in morbidly obese patients (31).
Additionally, within the 20 associated regions, we confirmed two previously published obesity associations: CNVR 1p31.1 20 kb upstream of NEGR1 (7,8) and CNVR 10q11.22 (1) covering the four genes SYT15, GPRIN2, LOC728643 and PPYR1 (19) with PPYR1 being the most interesting candidate, given its role in energy homeostasis and regulation of food intake (27,32). PPYR1 null animals have, for instance, a reduced body weight. PP reduces food intake predominantly via stimulation of the anorexigenic melanocortinergic pathway. This effect is mediated by direct action on local PPYR1 within the arcuate nucleus (32). PP binds to the PPYR1. In our study as well as in the previous study which initially described the association of CNVR 10p11.22 (1) with BMI (19), the loss of a PPYR1 gene copy number was associated with obesity. Among those individuals (n = 57) of our two GWAS discovery samples that were called for the loss of a PPYR1 gene copy number, only one was homozygous for the deletion. One can only speculate about these seemingly contradictory findings between studies in human and mice. Several mechanisms might explain the difference: (i) mice may not be the most appropriate model to understand the function of the human PPYR1, as mice express a functional Y6 receptor (in humans only an non-functional pseudo gene exists) and this receptor could explain some of the results of the Ppyr1 knockout mice. (ii) Sequence identity between mice Ppyr1 amino acid sequence is only 76% compared with human PPYR1. The high variability of PPYR1 across species might explain its different roles among species. (iii) Ppyr1 knockout mice are hyperphagic but nonetheless lean (27). If the gene has a slightly altered function in humans, hyperphagia is potentially retained but not the lean phenotype. (iv) Compared with the 56 studied subjects who are hemizygous for the PPYR1 gene, Ppyr1 knockout mice have neither copy of the gene nor corresponding gene expression products. Thus, subjects with only one copy of PPYR1 may produce less protein, which will regulate energy homeostasis through agonists or other pathways to inhibit obesity. In contrast, knockout mice have neither copy of the gene nor the corresponding gene expression products, which may also explain the difference between human studies and mice experiments.
While the replications of previous findings underline the strength of our study, the focus on consistently associated common CNVs for early-onset (extreme) obesity using an agnostic genome-wide approach is also accompanied by weaknesses. First of all, our follow-up/replication step using tagging SNPs has focused on only 2 of all 20 candidate CNVRs-albeit those with the strongest association signals. Thus, it is conceivable that additional regions could be true positive findings. However, given their weaker association signals in both of our deletion samples, larger replication samples than the one used by us will be required to rule out false-negative findings. This is underlined by the results of our array-based replication analyses supporting four additional CNVRs of all 20 candidate CNVRs. Secondly, our study clearly has limited detection power to comprehensively assess CNV at a genome-wide level. None of the tested CNVs achieved a stringent genome-wide significance level and for those that could be replicated only moderate effects (odds ratio of ~1.2) were observable. Thus, our observation for early-onset (extreme) obesity underlines the recent finding that common CNVs are unlikely candidates to explain larger parts of the 'missing heritability' (26). Finally, it has to be underlined that the focus on CNVRs is accompanied by (a) the difficulty to define the CNV markers that make up a CNV and (b) the difficulty to assess when and if association signals in different samples pertain to the same source. Here both problems have been dealt with by providing upper limits of correlations among associated CNVs of independent samples and by omitting combined P-values across samples.
Since we replicated two CNVRs using tagging SNPs, their association could have already been detected by genome-wide SNP association studies. Indeed, rs2815752 (tagging CNVR 1p31.1) near NEGR1 is a well-known and well-replicated BMI locus (8,33). In contrast, for rs9804659 (tagging CNVR 11q11), no association to obesity, BMI or waist–hip ratio has yet been reported in the primarily population-based genetic association studies of adults (33,34).
Very recently, another 21-kb CNV that lies 50 kb upstream of the gene GPRC5B has been reported to be associated with BMI (33). The identification of this CNV was based on the tagging SNP rs12444979 in a large meta-analysis for population-based samples, whereas the deletion allele of the CNV is tagged by the non-risk allele of the SNP. We could also identify this CNV as one of the 244 common CNVRs analysed here. However, a significant association with early-onset (extreme) obesity was observed only in our case–control GWAS discovery sample (one-sided min. PCC = 0.0066 and min. Pfamily-based = 0.0408 for the non-deletion direction).
Owing to our focus on common CNVs, the role of rare deletions or duplications has only been addressed here, by exploring selected loci harbouring known rare CNVs in extreme obese patients (20,21). In order to comprehensively investigate whether certain rare CNVs [e.g. large (>1 Mb) CNVs (cf. 22)] predispose to obesity, a more comprehensive quality control (QC) and validation of each single CNV call as well as a much larger adequately powered analysis sample is needed. It is conceivable that such assessments will become feasible within large-scale consortia such as GIANT [Genetic Investigation of ANthropometric Traits (8)].
In summary, we provide evidence for a new common CNVR for early-onset (extreme) obesity on chromosome 11q11 covering the coding regions of OLR genes. Moreover, we confirmed two previously described common CNVRs for obesity. Overall, however, common CNVRs seem to be of minor importance to account for the 'missing heritability' of early-onset (extreme) obesity.
Detection of common CNVRs and initial association analyses were based on two GWAS discovery samples both genotyped by the Affymetrix Genome-Wide Human SNP Array 6.0. The family-based GWAS sample (Sample 1) consists of 424 obesity trios, i.e. one (extremely) obese child or adolescent and both biological parents (Supplementary Material, Table S1). The second GWAS sample (Sample 2) consists of 453 (extremely) obese children and adolescents and 435 normal-weight or lean adult controls (Supplementary Material, Table S1). For replication of the association of certain CNVs with obesity as derived from both GWAS samples, we analysed a third independent sample of 365 obesity trios (Sample 3) which was recruited similarly as the family-based GWAS sample (Supplementary Material, Table S1). Additional array-based replication analyses were performed in a subsample of Sample 3 consisting of 281 obesity trios for which genome-wide genotyping data (Affymetrix Genome-Wide Human SNP Array 6.0) became available very recently (Sample 3a).
In all three samples, the measured BMI (in kg/m2) was assessed for extremeness using age- and sex-specific percentile criteria for the German population (35). According to this reference population, all cases (cases of the case–control sample and offspring of the two family-based samples) were at least overweight (percentile ≥ 90th). Most of the cases were extremely obese (percentile ≥ 97th): 93.6% of the index cases of the family-based GWAS sample, 92.5% of the cases in the case–control GWAS sample as well as 91.5% of the offspring from the family-based replication sample.
Written informed consent was given by all participants and, in case of minors, by their parents. The study was approved by the Ethics Committees of the Universities of Marburg and Essen and conducted in accordance with the Declaration of Helsinki.
In both GWAS discovery samples, genotyping was carried out using the Affymetrix Genome-Wide Human SNP Array 6.0. Besides 906 703 SNP probes (869 747 autosomal), this chip contains 943 390 non-polymorphic additional probes (888 023 autosomal) for copy number analyses (http://www.affymetrix.com). The autosomal fluorescent intensities at the non-polymorphic probes were used as a measure of copy number variance for the primary statistical analysis. This was done to avoid the loss of information and power related to CNV calling (36,37). The intensity values were extracted from the individual CEL files utilizing the R-package 'affxparser'. We normalized the raw intensities of the case–control GWAS sample by performing quantile normalization (38) to address potential chip effects. In case of the family-based GWAS sample, we analysed the raw fluorescent intensities since the family-based design allows for controlling the inter-individual variability.
As secondary sensitivity analysis in both GWAS discovery samples, we investigated the called CNV states at a genome-wide level, i.e. the unphased total number of copies at a genomic locus. We determined CNV calls for each individual and each marker using the Affymetrix Genotyping Console 3.0. In particular, we applied a five-state hidden Markov model (HMM) (39) to smooth and segment the data with default values of 0.2 for each prior probability and 1000 Mb as a transition decay. The five possible HMM copy number states (CNS) were: 0, homozygous deletions; 1, heterozygous deletions; 2, (normal) diploid case; 3, single copy gains; 4+, cases of four or more copies. The CNV calling requires comparison of individual signal intensities against a reference sample. Owing to computational constraints, we processed a reference sample of 106 parental pairs from the family-based GWAS sample. To check whether and to what extent the choice of the reference sample had an impact on CNS calling, we decided to choose two different reference samples, each comprising 106 parental pairs. Reference Sample 1 was a random sample of all parental pairs, whereas reference Sample 2 was based on those 106 parental pairs with the lowest mean BMI standard deviation score values out of all non-obese parental pairs (percentiles < 90th) in our sample (see Supplementary Material, Table S1 for details). In both GWAS discovery samples, we performed CNV calling based on either of the two reference samples and proceeded only those variations that were consistently assigned via both approaches (see QC and association testing section).
A CNV has been defined as a DNA segment that is 1 kb or larger and is present at variable copy numbers in comparison with a reference genome (40). Copy number variable regions (CNVRs) are regions covered by overlapping CNVs (41). The copy number characterization of 270 HapMap samples showed that ~80% of observed copy number differences between pairs of individuals were due to common CNVs with an allele frequency of >5%, and >99% were derived from inheritance rather than from new duplication/deletion events (13). According to published recommendations (13,41), we defined common CNVRs as regions covering at least three consecutive CNV markers showing deletions or duplications in at least 5% of each set of GWAS cases, parents and controls. Thus, if several individual CNVs overlapped, we merged them in the same CNVR, so that most CNVRs represent a union of overlapping CNVs (19,42). For details on the resulting 244 CNVRs based on 8051 CNV markers with genomic positions based on genome build hg 18 (March 2006), see Table 1 and Figure 2.
The most promising new region detected in the two GWAS samples, a CNVR at chromosome 11q11 (CNVR 11q11), was technically validated in the family-based GWAS sample using qPCR to ensure reliability of the chip-based CNVR detection and for additional analyses based on precise individual copy number calls. In more detail, we applied real-time quantitative PCR, using a Duplex TaqMan CNV assay (Applied Biosystems, Germany; assay Hs03802074_cn at chr11:55 203 791 ± 50 bp) as described previously (43). Briefly, every reaction was performed as a triplicate and the results for the qPCR were analysed using the software CopyCaller 1.0 (Applied Biosystems). Overall, the correlation between continuous intensity-based CNV calls used for association testing and continuous qPCR predicted calls was estimated to be 0.87 (Pearson correlation coefficient). Using qPCR approximately half of the individuals of the family-based sample were identified as carriers of a heterozygous or homozygous deletion at CNVR 11q11. Regarding called CNS the data were not readily comparable. Hence, all called 105 homozygous deletions at CNVR 11q11 were called by both qPCR and Affymetrix CNV markers whereas the overlap for called heterozygous deletions (n = 510 called by qPCR) was exceedingly lower (only 3% of all 510 heterozygous deletions were calculated as CNS = 1 by the HMM applied to the Affymetrix CNV markers). Taken together, the latter observation corresponds to the results of others (44) and underlines the benefits of using intensities as compared to the use of called CNVs.
For further analysis of the CNVR 11q11 signal, in the independent sample of 365 obesity trios copy number status was also assessed by the qPCR assay described above, resulting in similar proportions for heterozygous and homozygous deletion carriers.
CNVRs and the surrounding ± 500 kb DNA segments with evidence for an association to obesity as based on the CNV-FBAT (family-based GWAS sample; at least one marker with a two-sided asymptotic P-value ≤ 0.05) and with additional evidence for association as based on the logistic regression (case-control GWAS sample; at least one marker with a directionally consistent two-sided asymptotic P-value ≤ 0.05), were analysed with the aim to identify SNPs that are in linkage disequilibrium with the involved CNVs (see 26). It was reported that CNVs that were well genotyped were frequently tagged by SNPs (26). Again circumventing the calling, we determined Pearson correlations of intensity values for CNVs and SNPs in the set of all 424 parental pairs of the GWAS trios. For a CNVR-tagging SNP we required that the squared Pearson correlation coefficient (r2) of intensities had to be >0.6 for at least three consecutive CNV markers. CNVR-tagging SNPs were detected for two of the 20 CNVRs assessed. These two SNPs (rs9804659, rs2815752) were followed-up for replication association analyses in the sample of 365 independent case-parents obesity trios.
All individuals of both GWAS samples underwent SNP marker QC as described previously (4,45). In detail, QC filters for individuals were checked for Mendelian inconsistencies in the family-based GWAS sample (>5% of all SNPs), the application of an SNP call rate of 95% and a minimum genotyping quality as assessed by Affymetrix Contrast QC > 0.4.
Regarding QC of the CNVR determination, we limited our analyses to CNVRs for which the genomic variability-irrespective of phenotype (i.e. in cases, controls, parents and offspring)-was at least 5% based on each of the two reference samples used for copy number calling (Supplementary Material, Table S1). The mean individual concordance rates between the use of both reference samples for called CNSs 0, 1, 3 and 4+ were 97.83% (range: 84.67–99.90%) in the family-based GWAS sample and 94.53% (range: 83.61–99.79%) in the case–control GWAS sample.
Concerning the genome-wide agreement between CNVR calling in both discovery GWAS samples, a total of 880 CNVRs with frequencies >1% which cover ≈15% of the genome have been called in the family-based discovery GWAS sample. Of these CNVRs, 97.04% were again called as CNVRs in the case–control discovery GWAS sample. Even among those CNVRs that were found in at least two individuals (6473) in the family-based discovery GWAS sample, 83.5% were redetected as CNVRs in the case–control discovery GWAS sample. CNVRs called in just one individual are more likely false positives compared with CNVRs identified in several individuals and are thus neglected here.
When focusing on CNVRs, comparability of association findings across samples needs to be addressed as different individual CNV markers might contribute to the association in different samples. We determined pairwise Pearson correlations between pairs of selected CNV marker intensity values in each of the two discovery GWAS samples. The selection was based on the presence of some evidence for an association with obesity for the CNVR (family-based GWAS: two-sided asymptotic P-value in CNV-FBAT ≤ 0.05; case–control GWAS: directionally consistent two-sided asymptotic P-value in logistic regression ≤ 0.05). We report the maximal Pearson correlation coefficient (r) computed from the set of selected CNV markers (Table 2, 10th column). For comparability, we also report the Pearson correlation coefficient (r) for those two CNV markers with minimal P-values in the family-based and in the case–control discovery GWAS samples, respectively (Table 2, 9th column).
Concerning the QC related to the qPCR experiments for CNVR 11q11, only those copy number calls with a confidence value (by CopyCaller 1.0) of at least 0.98 without evidence for Mendelian inconsistencies were followed-up. Subsequently, the full set of all 772 families successfully assessed by qPCR was analysed for deviations from Hardy–Weinberg equilibrium (46) resulting in a two-sided exact P-value of 0.95 in the parents.
For QC of the CNVR-tagging SNP genotypes (at rs9804659 and rs2815752) in the replication sample of 365 case-parents obesity trios, an exact test for deviations from Hardy–Weinberg equilibrium (46) was performed in the set of parents (minimal two-sided exact P-value across both SNPs = 0.33).
In each sample, we analysed the association between CNVs and (extreme) obesity. All primary analyses of the GWAS discovery samples were based on fluorescent intensities which represent the copy numbers. For the family-based GWAS sample, we applied the CNV-FBAT methodology (37) to test the raw intensities for association with the phenotype obesity. We based our association tests on the raw intensities in order to avoid the known loss of information and statistical power resulting from classifying raw copy number measurements into discrete classes, i.e. CNV calls (36,37). To reduce the number of statistical tests and to minimize the risk of false-positive findings, the CNV-FBAT association tests were limited to the common CNVRs derived as described above. Additionally, we performed multi-marker FBATs (35) incorporating all possible marker combinations in a specific CNVR. The case–control GWAS sample was analysed by logistic regression with the predictors normalized intensities, sex and age.
Regarding the study design we focused on CNV testing in the family-based discovery sample as a first step while using the case–control discovery GWAS sample as secondary discovery filter. The focus on our family-based sample has several advantages: (i) the known robustness of the family-based design against population stratification; (ii) better control for biases (e.g. plate effects) due to the within-family comparisons; (iii) due to the incorporation of only one affected offspring per pedigree, the CNV-FBAT leads to quite conservative results, which lowers the risk of false positives. The conservativeness of the CNV-FBAT is displayed in the QQ plots for the two discovery samples (Supplementary Material, Fig. S2). In contrast to the CNV-FBAT, the results for the case–control analyses are too liberal although we performed normalizations of the intensity signals to, for example, address technical problems (e.g. plate effects).
To derive CNVRs for replication using CNVR-tagging SNPs, we first focused on those estimated CNVRs which showed some evidence for association to obesity in the family-based GWAS sample (CNV-FBAT test; at least one marker with a two-sided asymptotic P-value ≤ 0.05). Taking into account whether this CNVR indicated gain or loss for the obesity association, we then calculated CNV marker-specific one-sided P-values for the tests in the case–control GWAS sample. For clarity (if not stated otherwise), all reported P-values are one-sided, nominal, i.e. unadjusted for multiple testing, and asymptotic.
In the independent replication sample of obesity trios, we genotyped the two identified CNVR-tagging SNPs. For detailed analysis of the CNVR 11q11, this was additionally genotyped by qPCR in the family-based replication sample. For the CNVR-tagging SNPs, we applied the classical transmission disequilibrium test (TDT) (47). Replication analysis of the qPCR-typed CNVR 11q11 was performed by application of the CNV-FBAT on the derived copy number calls. Odds ratios (ORs) were calculated by applying the haplotype relative risk method (48), which uses the transmitted parental alleles as a case sample and the untransmitted parental alleles as a control sample.
For additional array-based replication analyses of the 20 discovered CNVRs in a sample of 281 independent obesity trios, intensity values for each involved marker were again extracted with the R-package 'affxparser'. As for the family-based discovery GWAS sample, the CNV-FBAT methodology was applied to the raw intensities afterwards. Finally, one-sided P-values were calculated by taking into account the estimated CNVR effect direction as based on the family-based discovery GWAS sample.
Under the null hypothesis of no marker-phenotype associations, the intensity distribution at each marker is independent of the family status (i.e. father, mother, child) in the family-based samples and independent of the affection status (i.e. case, control) in the case–control sample. Thus, in order to simulate the test statistics distributions under the null hypothesis, we permuted family status (and family labels) or affection status, respectively, several times; each time storing the corresponding test statistics. Following this procedure, marker-wise approximate exact P-values were then calculated by dividing the number of observed permutations achieving an equal or higher test statistic for this marker than the actually observed one by the number of permutations performed. The QQ plots in Supplementary Material, Figure S3 demonstrate the validity of this approach and of the asymptotical P-values by comparing the quantiles of the 8051 true (asymptotic) observed P-values in the 244 common CNVRs with those quantiles of approximate exact P-values based on 1000 permutations in the case–control sample and the family-based sample, respectively. Note that here, deviations from the diagonal line for the case–control sample result from the limited number of permutations performed. CNVR-wise approximate exact (called CNVR-adjusted) P-values were calculated analogously by taking into account that several CNV markers have been tested for each CNVR. Thus here, for each permutation and each CNVR, the maximum over all CNV markers test statistics was compared with the maximum of the observed test statistic. In order to achieve one-sided exact P-values, we additionally stored and compared the direction of the effect.
To address the genome-wide multiple testing problem of our report, we also performed additional analyses. First, to address the impact of the 25 CNVRs detected in the family-based GWAS discovery sample, we stored the CNV-FBAT P-values of the respective 1314 CNV markers (those with two-sided asymptotic P-values ≤ 0.05; Table 2). Next, we drew 1000 random sets of 1314 CNV markers out of the 244 analysed CNVRs (8051 CNV markers) to derive the distribution of P-values under the null hypothesis. Next, both the quantiles of –log10 P-values of observed association signals and the corresponding 97.5, 50 and 2.5% percentiles of the 1000 random sets of equal size were plotted against the expected quantiles (from a uniform distribution) in a QQ plot (Supplementary Material, Fig. S1). While both the observed and sampled results indicated the conservativeness of the family-based discovery GWAS approach, the smaller P-values of the observed signals justify our subsequent proceeding.
Secondly, in the case–control GWAS discovery sample, we performed 10 000 permutations of the case–control status to see how often we get directionally consistent nominal minimal one-sided P-values of ≤0.025 as observed for 20 out of the 25 CNVRs detected in the family-based discovery GWAS sample. In none of the permutations we observed more than 20 such directionally consistent CNVR findings; in two permutation samples, we observed exactly 20 (taken together resulting in an empirical P-value of 2 × 10−4); the median was 10 directionally consistent CNVR findings in the 10 000 permutations.
Thirdly, in order to account for multiple testing in the case–control GWAS discovery sample, we also performed Bonferroni corrections for the 25 CNVRs tested. Five of the 25 CNVRs followed-up in the case–control GWAS discovery sample achieved one-sided P-values of <0.025.
OR4P4; HGNC: 15180, Entrez Gene: 81300, Ensembl: ENSG00000181927, UniProtKB: Q8NGL7.
OR4S2; HGNC: 15183, Entrez Gene: 219431, Ensembl: ENSG00000174982, UniProtKB: Q8NH73.
OR4C6; HGNC: 14743, Entrez Gene: 219432, Ensembl: ENSG00000181903, UniProtKB: Q8NH72.
I.J., J.H., A.H., A.S. conceived and designed the experiments, C.V. performed the replication and validation experiments, I.J. analysed the data, J.H., A.H., S.S., H.S. contributed to reagents/materials/analysis tools and I.J., J.H., A.H., A.S. wrote the manuscript. All others reviewed and approved the manuscript.
This study was supported by grants of the German Federal Ministry of Education and Research (BMBF 01KU0903; NGFNplus: 01GS0830, 01GS0820). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding to pay the Open Access publication charges for this article was provided by grants of the German Federal Ministry of Education and Research, NGFNplus: 01GS0830.
Supplementary Material is available at HMG online.
We thank the probands and their families for their participation and support.
Conflict of Interest statement. None declared.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Puppy stomped to death, thrown in dumpster
Christopher Blackstone's mug. Larger image below.
Two days before Christmas, Christopher Blackstone of Colorado Springs got into an argument with his girlfriend, Talitha Peterson-Hooks. She ignored his subsequent calls and texts, and, according to the police report, that's when Blackstone turned to Peterson-Hooks's three-to-five-month-old puppy and stomped on its head repeatedly.
Early Christmas Eve morning, Peterson-Hooks got a text message from Blackstone that read, "If you would have answered your phone, your dog would still be alive." He told her to check the dumpster behind their apartment. She found her puppy spattered with blood in a white trash bag and called the police.
According to the Colorado Springs Police Department, animal cruelty cases this extreme are rare in the city. "It pops its head up once in a while," says Sgt. Darrin Abbink. "But it's not a common thing." On the other hand, Abbink says it is relatively common to see domestic violence cases that involve the victim's animals. "It's just one method of control that apparently this individual resorted to," he says.
Blackstone, who has a history of domestic violence, has been accused of felony cruelty to animals. When he was arrested, he tried to kick his way out of the patrol car, an act that earned him a felony charge for criminal mischief.
The Humane Society just released its annual Humane State Rankings, and Colorado came in a highly respectable fifth, scoring a 38 out of 65. So this is a pretty alright state to be in if you're an animal, as long as you're nowhere near Christopher Blackstone.
Christopher Blackstone's booking photo. Courtesy of the Colorado Springs Police Department.
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Hundreds of thousands of people attend Anzac Day dawn services at Gallipoli, Villers-Bretonneux and across Australia to mark exactly 100 years since Australian and New Zealand troops came ashore.
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An engineering graduate from the Australian Defence Force Academy creates a microscopic, but no less significant, Anzac Day tribute on a piece of gold much smaller than a pin head.
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XTracPads Fat Mat
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Advertised as the mouse mat for Professional gamers, the new XTracPads Fat Mat gets some OC3D lab's lovin'.
CableMod Launches Cooler Master Series PSU Kits
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Today CableMod is proud to launch CM-Series Premium Cable Kits.
Alphacool GPU Cooling Configurator
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Alphacool have just released their new GPU Cooling Configurator tool, so that users can more easily find a compatible NexXxos GPX Water Block.
EU opens Antitrust Investigations Against Qualcomm
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The European Commission has opened two formal antitrust investigations into Qualcomm.
Microsoft Surface Pro 4 Tablet to Contain Intel Skylake and a NVMe SSD
Mark Campbell | 16th July 2015
Rumors suggest that Microsoft's Surface Pro 4 Tablet will Contain an Intel Skylake and a NVMe SSD.
World of Tanks 9.9 update brings new events and graphical improvements
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Mark Campbell | 16th July 2015
The Retail version of Windows 10 will be releasing on August 16th and will come on USB stick.


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Photo Gallery
New health plans may leave some 'underinsured'
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This photo of part of the HealthCare.gov website is photographed in Washington, in this Nov. 29, 2013 file photo. Newly released federal figures, as of Nov. 30, 2013, show more people are picking private insurance plans or being routed to Medicaid programs in states with Democratic leaders who have fully embraced the federal health care law than in states where Republican elected officials have derisively rejected what they call "Obamacare."
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1000+ Rentals · Barcelona
1000+ Rentals · Barcelona
Ideally located in Barcelona's city center, the SUPER CENTRAL DOUBLE ROOM boasts natural lighting and a comfortable space, with a big closet. Only 5 minutes by foot from Plaça Catalunya, Gotic neighborhood and 6 metro lines. Learn More
The apartment has three rooms. This includes the room advertised here, another double room which I also rent sometimes through Airbnb (thus you may coincide with other guests in my home), and a single room where I live. This double room has its own closet, and there is a bathroom in the hallway. You also have access to the bright and airy lounge room, a large southwest-facing terrace, and the kitchen. This is a friendly and international apartment welcoming solo travelers, couples (gay-fri...
El Gòtic, Barcelona
Mi piso es perfecto si buscas poder dormir tranquilamente, se encuentra en zona residencial. Muy bien comunicado solo a 15 min en autobús del centro. Yo vivo solo en el piso. Habitación de 9m2. cama individual, con armario, mesita de noche. El piso tiene 70 m2 en total, el comedor 18m2, terraza 5m2. Tiene mucha luz exterior. Todas las ventanas dan el exterior. tendrás acceso a todo el piso, cocina baño, comedor, terraza. Yo vivo en el piso, así que si necesitáis ayuda de cualquier tipo est...
Sants-Montjuïc, Barcelona
We are ready to share with you our house in the hearth of Barcelona, in the Gotic Area, close to the Ramblas and most of the attractions, facilities, restaurants, bars, shopping streets, point of interest and much more. Learn More
SINGLE ROOM available in our house. The apartament was recently renewed and is located in an old 150 years old building, my wife personally designed the house concept and we're sure that you will enjoy it! Our guest will have a private room and the toilet only shared with the other guests. We live in one side of the house and the big living room is shared with everyone, but you will have your own privacy and no problem if you want to have some relax on the sofa with your computer. Toilet...
El Gòtic, Barcelona
Single room in the center of the most important monuments of Gaudi. Learn More
Double room interior . In a beautiful stately apartment . this against the L3 and L5 " Verdaguer " metro station. Located in one of the best areas of Barcelona, the Eixample Dret , where the best architectures such as Gaudi 's Sagrada Familia , La Pedrera and Casa Batllo , Casa de las Puxes & 5 min walk to the emblematic district of Gracia . The area is very safe for walking . It is a very well connected with metro stations , trains and buses ; and a variety of restaurants of all va...
Dreta de l'Eixample, Barcelona
This is a unique apartment of 60m2 in the center of Barcelona. 2 min from las Ramblas and Plaza Catalunya. 5 minutes from MACBA (Museum of Modern Art) and Boqueria Market. FREE WIFI Learn More
This is a unique apartment of 60m2 in the center of Barcelona. It has a large bedroom with a double bed (160x200), a spacious bathroom, a full equipped kitchen and living room with flat led TV and DVD. In the building there is no elevator ... so a little bit of exercise to climb 4 floors !!! SERVICES INCLUDED: - Free wifi - Bed Linen - Towels - Hairdryer - Toiletries (toilet paper, shower gel, shampoo, toothpaste ...) - Tea and coffee - Fridge with bottled water and soft drinks, ja...
El Raval, Barcelona
Ofrecemos 1 habitación (o 2) en nuestro increíble apartamento en el centro de Barcelona. Tiene sofá cama, cajonera y puertas al exterior. El edificio refleja la arquitectura clásica de Barcelona pero con el interior totalmente reformado y moderno. La fachada de la finca es antigua y clásica aunque el interior está totalmente reformado. La decoración es algo peculiar puesto que hemos fusionado el estilo minimalista con lo vintage. En nuestro hogar disponen dos habitaciones, una de ellas doble...
Sant Pere/Santa Caterina, Barcelona
Unique experience in stylish vintage flat in the heart of Gotico. Quiet room, charming retro environment in Barcelona's most desirable area, Sant Jaume, the city's emblematic square. Walking distance from Ramblas and most sites of interest. The flat is a vintage delight. Breakfast room, reading room, dining and sitting room have all been preserved with original fittings. Balconies overlook a lively square -perfect setting for a relaxing evening aperitif- and bedrooms to the inside f...
El Gòtic, Barcelona
Sleeps Max: 2 •Gothic District, city center Barcelona. •1 Double bed •2 Bathroom •2 Lounges (one with balcony) • kitchen with pantry • a balcony Learn More
The apartment has more than 140 square meters, where you will feel at home. Each guest can come and go when they want at the apartment and the last day you can leave your luggage on it Housekeeping, sheets and towels. The apartment is perfectly located. Less than 3 minutes Walking to Plaza Real, Las Ramblas, Barcelona Cathedral, Church of Santa Maria del Pi. Less than five minutes walk from Paseo Colon, the port of Barcelona, Palau de la Música, Plaza de Catalunya. About ten minutes walk...
El Gòtic, Barcelona
Sofa bed in a large living room in an 65m2 bright apt. with a terrace and bbq on the rooftop, located in the city center, in the trendy Raval Alto hood near La Rambla. Absolutely perfect for small budget travellers :) Learn More
Super comfy sofa bed in a large living room. 65m2 flat, very bright and ideally located. Private rooftop terrace with a bbq, big table and deck chair, perfect for a breakfast under the sun or a dinner with the sunset. Ideal for small budget travellers! Located in the heart of the old city (Ciutat Vella), in the trendy nieghborhood of Raval Alto. 5 minutes walk to La Rambla, 10 minutes walk to the Cathedral and the Gotico, 15 minutes walk to the sea and Port Vell (marina). The flat co...
El Raval, Barcelona
Sunny room in a family house in a unique place in Barcelona, private bathroom, air conditioning in the room. AC WI-FI TOWELS BREAKFAST Learn More
Beautiful large room in Montjuic ParkThe apartment is located in the Pasagge Martres, near the Greek Theatre in Montjuic which is one of the areas with the most beautiful gardens of Barcelona. Room: Is broad, clear with a nice view onto an interior garden. You have the option of a double or twin beds and the room has private bathroom. Towels and sheets are provided. Wi-Fi free. There is the possibility of accommodating a third person in the room.In the apartment you can have space in the ...
Sants-Montjuïc, Barcelona
Welcome to the oldest part of the city. The refurnished flat is really very spacious with high ceilings so you will feel home. All interesting spots/public transportation are within walking distance ;-) Unique **** bedroom/suite complete with dressing and private bathroom in the old heart of Barcelona. The room is really very large with high ceilings , guests are always impressed, even after they'd seen the pictures previously. Your own bathroom is at the end of the corridor (like in most...
El Gòtic, Barcelona
Double bed and private bath room with capacity to comfortably accommodate 2 or 3 people. The apartment has 90 m2 balcony, kitchen and living room of 30m2 blend comfort and functionality, with minimalist furniture and a very cool and casual design. Learn More
Our home is a big place, with good light, the sun comes and goes for all the different rooms during the day. The common zones and the rooms are very comfortables and cozy. Is located in a central place which let the people know and see the city in short time due to the easy access. The kitchen is equipped with stove, oven, microwave, refrigerator, freezer. The guest can use a drawer to store things and a refrigerator compartment, plus the use of the washer and dryer is allowed. The room ...
Dreta de l'Eixample, Barcelona
The apartment is located in a great area of Barcelona's Raval . The apartment is fully ready to receive guests. Learn More
Apartment vividly expresses the culture of everyday life of the last century. The old floor tiles, stained-glass windows in the doors, beamed ceiling, balcony doors with shutters and left the kitchen as a decoration oven doors Wet hollowed out of a piece of marble, it all interested fans to plunge into the atmosphere of the past. Anyone tired of the same type of hotel rooms, which are identical and soulless. Immerse yourself in the comfort and relax the soul in the heart of Barcelona! Rava...
El Raval, Barcelona
La habitación a alquilar de unos 14 m2 consta de 2 ventanas todas ellas al exterior, acogedora equipada con calefacción, ventilador de techo, 2 armarios, caja fuerte y un baño privado con ducha. Disponen de llave para cerrar puerta de la hab.
Torre Baró, Barcelona
Single bedroom for rent in renovated apartment with luxury services. 200m from the Sagrada Familia, 50m from the Metro, at the intersection of Castillejos Street and Avenue Gaudí Learn More
La Sagrada Família, Barcelona
estudio con mucho encanto , restaurado , con ascensor .suelo de tarima de madera . en un solo ambiente . bañera abierta . muy romantico . mucho sol . pequeña terraza con una mesa y dos sillas si esta ocupado hay uno enfrente 712779 estudio de 45 mts , muy romantico , con una bañera incorporada , dos balcones al patio interior del ensanche , mucho sol , pequeña terraza , suelo de tarima de madera y vigas originales rodeado de tiendas , bares de tapas , restaurantes ... esta situado e...
L'Antiga Esquerra de l'Eixample, Barcelona
Sleeps Max: 2 •Gothic District, city center Barcelona. •1 Double bed •2 Bathrooms • A lounge (with balcony) • Kitchen with a pantry • Balconies Learn More
The apartment is large and bright with more than 140 square meters, where you will feel at home. Each guest can come and go as they please. On your last day we are happy to hold your luggage until you depart. .Our guests have acces to all commom areas of the house, including the kitchen, the living room, balconies, and 2 bathrooms. We hope you will feel at home in our home. We know Barcelona very well and will provide you with the help that you need to have a great vacation in our beauti...
El Gòtic, Barcelona
Our lovely flat is very spacious and with nice people. Nice big room perfect for 2 people. Apartment fully equiped and very centric (Gothic quarter) 10 min by walk from plaza Cataluña and 5 min from the famous gothic cathedral. Close to the beach Learn More
El alojamiento Tenemos un apartamento muy amplio y agradable. Es muy agradable estar en el balcón, donde verás una calle bonita, con árboles, la gente paseando con sus mascotas y sentir la energía de la ciudad. La cocina es equipada, nuestros huéspedes tendrán un sitio en la nevera y también un espacio en el armario para dejar sus cosas. La lavadora se puede utilizar a cada 3 días, tenemos una terraza que se utiliza como tendedero. El baño es compartido y la limpieza hacemos diariamente,...
El Gòtic, Barcelona
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By Floyd Ingram Chickasaw Journal HOULKA – Houlka is the perfect place to grow up and also the perfect place to raise a family and Terri Turner has done both. Turner was named the 2015 Chickasaw County Woman of the Year and it doesn't take long to realize why people chose this Chickasaw County […]
Tag Archives | Terri Turner
TERRI TURNER: 2015 Woman Of The Year
By Floyd Ingram | 7:05 AM | June 28, 2015 | Living, News
"Giving hunger a rest"
By Floyd Ingram | 7:05 AM | March 30, 2014 | Business, Living, News
HOUSTON – Franklin Corporation entered the United Way of Northeast Mississippi competition with a food sculpture representing what it does best, by building a recliner that moves. Companies across Northeast Mississippi were asked to contribute to the United Way "United Against Hunger" food drive to celebrate a record-breaking year and to provide food […]
- DANIEL GARDNER: Love and Hate July 22, 2015
- Area students earn ICC scholarships July 26, 2015
- Houlka trustees Ok demolition, infrastructure details July 27, 2015
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- ICC offering newNational Guard program July 27, 2015
- Victoria Wofford awarded Yates Scholarship July 29, 2015
- Suspicious mailers sent before Primary Election July 29, 2015
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- Area residents graduate ICC program July 28, 2015
- Area trio earns ICC scholarships July 28, 2015
- Jimmie Hardin: I for one cared that it was a week late. I live in...
- Daniel Drown: Great article about this timeless masterpiece that...
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text: The GOP's Latest War On Family Planning ByLauren RankinPublishedJune 23, 2015, 6:00 AM EDT Yet again, House Republicans are taking aim at family planning. Last week, the House Appropriations Labor-HHS Subcommittee released a budget proposal for fiscal year 2016 that would remove all family planning funding under the Title X program. The Title X program provides access to comprehensive sexual and reproductive health care, including cancer screenings, contraceptive care, STI testing and much more, for 4.6 million people. The House GOP just declared war on it. Read More →


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Grantham Enthusiast
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- It's brilliant for water consumption as you don't realise how much water you have sucked up, due to the design of the bottle. Huge range of colours to meet everyone's taste.
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- The price is a bit excessive for what it actually is.
Published on: 03 August 2015


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DanLabGames has announced Creepy Mines 2, its latest game release for the Mac. Creepy Mines 2 is a not a standard classic brick game, but instead offers various new elements to make the gameplay "refreshing and new." The new game uses 3D graphics and has "some awesome effects." DanLabGames is offering a limited discount on the game for customers who bought Creepy Mines 1. Registration is $20 for new users.
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With primary residences you can generally get approved for an FHA loan with a score as low as 620. However, if this will be a second(vacation) home, or investment property your mid score generally has to be 680 and above for most lenders.
As you can see it is not straight forward, if you are working in NY and purchasing in NY, the home can be treated as your primary residence, and the scoe required and down payment are lower.
Besides a Realtor i am a NMLS Mortgage Loan Officer. I can be reached at 407 758 2731 to answer any questions you may still have.
Looks like your question has been answered. This area offers a very good value for the money. How did you decide on this area if I may ask.
Call me any time if you have any questions at 407-929-9674
Have a great day.
I agree with the above comments, the area of Avalon Park qualifies for USDA loans as well (100 % financing) I live in the area and yes that home is under contract and they had multiple offer situation, I will love to assist you, my office is located in Waterford Lakes and I know the area very well. There are several properties that are similar at the one in Sapphire Bay, Please feel free to contact me, I can also refer you to a local lender if is necessary.
Anabella Tercero Okrassa
Your Bilingual Realtor
(407) 797-5139
It depends on what type of loan that you were approved for.....if it were an FHA then at least 3.5% or Conventional then at least 20% of your offering price for the property.
James, III
RE/MAX Premier Properties
your mortgage pre-approval will tell you the details of the LTV (loan to value) ratio of your loan. For example, a conventional loan could have an 80% LTV, in which case you would be required to bring a 20% down payment. Your mortgage broker is also the only person who can tell you if this product is usable to purchase in Florida.
Brahim Hanchi
The property you selected is no longer available. It is now under contract and pending.
To answer your questions. If the property in Florida is your second home, then you would need a down payment of 20% minimum. However, if it is your main residence, then you would only need 3.5%.
You must have a very good credit score to have been offered such a substantial loan and that would probably be the case in Florida as well.
Please contact me if you require any additional information.


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27-9511 - CliffCon Touchproof Right Angle Plug 3 Pole Mini Zc
ZC Series are available in a range of 2 pole to 8 pole plugs and sockets. Primarily
they are intended for low voltage and signal applications. Contacts are tin plated,
and other metal parts are brass or mazac, nickel plated.
Mfr Name: Cliff Electronic Components
Mfr Number: FC684223R
27-9512 - CliffCon Touchproof Right Angle Plug 4 Pole Mini Zc
Mfr Number: FC684224R
27-9514 - CliffCon Touchproof Right Angle Plug 6 Pole Mini Zc
Mfr Number: FC684226R
27-9515 - CliffCon Touchproof Right Angle Plug 7 Pole Mini Zc
Mfr Number: FC684227R
27-9526 - 4PC/L CliffCon Inline Connector
A range of 4-pole touchproof plug and socket power connectors which are available in Blue for low voltage and Red for high voltage. Only plugs and sockets of the same colour are compatible, so red plugs cannot be plugged into blue sockets and vice versa.
Mfr Number: FCR2065
27-9527 - 4PC/P/LV CliffCon 4 Pole Low Voltage Connector Plug
Mfr Number: FCR2066
27-9530 - 4PC/P90/HV CliffCon 4 Pole High Voltage Connector Plug
Mfr Number: FCR20673
27-9533 - 90 Deg CliffCon 4 Pole Adapter Allows 90 Deg PCB Mounting
Mfr Number: FCR6929
33-1771 - RG-6 Snap-N-Seal Connectors w/ Nut Seal
For big home theater and satellite installation jobs, buy the Snap-N-Seal connectors in packages of 50. Use with #33-1792 SNS installation tool.
Mfr Name: Thomas & Betts
Mfr Number: SNS1P6
101-042 - Metal 3.5mm Phone Plug
Handles are durable molded plastic or nickel-plated brass 1/4'' maximum cable Mono
Mfr Name: Switchcraft
Mfr Number: 39F748
27-210 - Chassis Mount Female Solder-On BNC Connector
BNC Type Connector
Chassis mount female
Minimum 10
Mfr Name: Distributed By MCM
Mfr Number: 27-210
27-9297 - Double Bubble™ Compression RCA Connector for RG6 Cable
The Double Bubble compression tool incorporates the dies into the design of the tool to mold the metal of the Double Bubble connector around the cable. This reduces the cost of the connector and elminiates the need for a compression sleeve for compression.
Mfr Name: Belden
Mfr Number: DB6RCAU
27-9469 - Airtight 3 Pole XLR Male PCB 90 Deg Connector
3-pin XLR connectors which are intended for applications such as active speakers, where it is detrimental to have air movement in and out of the enclosure. Gasket supplied to seal the socket flange against the enclosure. Gold plated contacts.
Mfr Number: CP30062
27-9513 - CliffCon Touchproof Right Angle Plug 5 Pole Mini Zc
Mfr Number: FC684225R
33-4190 - Plenum Rated T Type Connector for RG-6 Cable - Ten per Package
Use with plenum type RG-6 coaxial cable Use with #22-100 hex crimp tool Ten per package
Mfr Number: PL56CH
Great place to order. Always fast shipping and service. Customer service is great. Thank you MCM for your dedication.


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Film review: The Legend of Hercules
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"I declare to you that I love another; he's the reason I want to wake up in the morning, embrace the day. If your sword wound him and his face was unrecognisable to me, I will love him. If his tongue was cut from his throat, and I hadn't his voice to delight me, I would love him. And if you make only a memory of him, I would love him even more eternally. That's what you have to look forward to."
The Legend of Hercules has nothing to do with the legend of Hercules (or Herakles as the Greeks call him) as we know it from Greek mythology.
I remember when I went to the local library as a little boy and returned home with an enormous picture book about the 12 labours of Hercules. This picture book was so huge, when I was reading it nobody could see me.
In this picture book I read about Hercules slaying a lion of Nemea and the nine-headed serpent Hydra, catching a Erymanthian boar, cleaning the stables of Augeas by means of a river, retrieving the flesh-eating horses of Diomedes and abducting the hellhound Cerberus from the underworld.
In those days, these heroic feats appealed to me. They still do. But you won't notice much of that in this Greek tragedy. In other words, it's a sad affair.
Ultimately, it's a pathetic hodgepodge of themes from various movies. A little bit of Pompeii, which was also about a forbidden love between a princess and a slave who was forced to be a gladiator in Pompeii. The fight scenes were a weak duplicate of 300: Rise of an Empire and 300 itself. Especially because of the use of slow motion effects.
A comparison with Gladiator is also easily made. And the end is a lot like a children's matinee on Saturday afternoon with cartoons of mythological superheroes such as He-Man: Masters of the Universe.
This movie is bland, uninspired and surely not legendary. The only impressive item in this movie was the chest of Kellan Lutz, who played Hercules. Perhaps it can impress teenage girls who marvel while looking at these muscular males. I was less impressed than looking at a crispy fried tiny chicken on my plate during lunch.
In 1200 BC, King Amphitryon invades the kingdom of Argos. It comes to a fight between him and his rival King Galenus. After defeating Galenus, the kingdom Argos becomes part of Amphitryon's empire. He tells his wife Alcmene that this victory was for her. However, Alcmene is outraged by the thirst for power her husband displays. That night she asks Hera, wife of Zeus, for help. Hera finally appears and predicts that she will give birth to Hercules, son of Zeus.
Hercules, whose father calls him Alcides, is born and after 20 years has grown into a handsome muscular young man who is in love with the lovely Hebe. When they hear that Hebe will be engaged to Iphicles, Hercules' older brother, the two lovebirds try to flee. They are caught and Hercules is sent by his father on a expedition to Egypt, hoping he won't return alive.
Ultimately it appears to be a trap and the regiment is being ambushed. Hercules and the faithful Sotiris are the only two who remain. They are sold as slaves to fight as gladiators. Hercules is now doing everything to return to Tires to stop his older brother and beloved Hebe getting married.
In terms of originality, this story won't win a prize quickly. The special effects aren't that impressive either. The fight with a lion at the beginning of the movie looks promising for a few seconds, but the animated lion has the same appearance as the computerised images of Jumanji in 1985. So not really an improvement.
The gladiator fights captivated me a bit, yet it was a huge disappointment at the end. The fight where Hercules had to engage with six never-defeated gladiators was something I looked forward to. But it was as if Zeus himself came along and interfered, because at that moment a storm came up. The first thunderbolt distracted me for a moment and I needed to rewind the whole thing because the great heroic fight was already over.
For those who can't endure blood, don't worry because there is not a single drop of it in this film. I think they ran out of fake blood after filming 300: Rise of an Empire.
There were still a few things I had to chuckle about, or that annoyed me. The final battle with the lightning sword, which smacked down hundreds of soldiers, and the subsequent battle that Hercules fought with his father, were a bit exaggerated. It seemed like a Jean-Claude Van Damme scenario, where the hero gets beaten up terribly first before he claims victory.
The performances are also fairly limited and meaningless. Lutz looks frightfully muscled and Scott Adkins is a rather sad-looking king instead of a fearsome ruler.
Maybe it's the overdose of sandals movies I've seen lately, but I wasn't impressed with this Hercules remake. The only wow feeling I got was during the opening scene where the camera pops out of the water, swings over a battlefield with oncoming soldiers, then sways between the defending archers - a brilliant moment. But this was not maintained throughout the film. The rest is an accumulation of stale and insipid situations.
Hopefully, Hercules with Dwayne Johnson, which comes out later this year, will be far better.
My rating: 2/10
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