Comparative Analyses of QTLs Influencing Obesity and Metabolic Phenotypes in Pigs and Humans

The pig is a well-known animal model used to investigate genetic and mechanistic aspects of human disease biology. They are particularly useful in the context of obesity and metabolic diseases because other widely used models (e.g. mice) do not completely recapitulate key pathophysiological features associated with these diseases in humans. Therefore, we established a F2 pig resource population (n = 564) designed to elucidate the genetics underlying obesity and metabolic phenotypes. Segregation of obesity traits was ensured by using breeds highly divergent with respect to obesity traits in the parental generation. Several obesity and metabolic phenotypes were recorded (n = 35) from birth to slaughter (242 ± 48 days), including body composition determined at about two months of age (63 ± 10 days) via dual-energy x-ray absorptiometry (DXA) scanning. All pigs were genotyped using Illumina Porcine 60k SNP Beadchip and a combined linkage disequilibrium-linkage analysis was used to identify genome-wide significant associations for collected phenotypes. We identified 229 QTLs which associated with adiposity- and metabolic phenotypes at genome-wide significant levels. Subsequently comparative analyses were performed to identify the extent of overlap between previously identified QTLs in both humans and pigs. The combined analysis of a large number of obesity phenotypes has provided insight in the genetic architecture of the molecular mechanisms underlying these traits indicating that QTLs underlying similar phenotypes are clustered in the genome. Our analyses have further confirmed that genetic heterogeneity is an inherent characteristic of obesity traits most likely caused by segregation or fixation of different variants of the individual components belonging to cellular pathways in different populations. Several important genes previously associated to obesity in human studies, along with novel genes were identified. Altogether, this study provides novel insight that may further the current understanding of the molecular mechanisms underlying human obesity.     

Bivariate Genome-Wide Association Analyses Identified Genes with Pleiotropic Effects for Femoral Neck Bone Geometry and Age at Menarche

Femoral neck geometric parameters (FNGPs), which include cortical thickness (CT), periosteal diameter (W), buckling ratio (BR), cross-sectional area (CSA), and section modulus (Z), contribute to bone strength and may predict hip fracture risk. Age at menarche (AAM) is an important risk factor for osteoporosis and bone fractures in women. Some FNGPs are genetically correlated with AAM. In this study, we performed a bivariate genome-wide association study (GWAS) to identify new candidate genes responsible for both FNGPs and AAM. In the discovery stage, we tested 760,794 SNPs in 1,728 unrelated Caucasian subject, followed by replication analyses in independent samples of US Caucasians (with 501 subjects) and Chinese (with 826 subjects). We found six SNPs that were associated with FNGPs and AAM. These SNPs are located in three genes (i.e. NRCAM, IDS and LOC148145), suggesting these three genes may co-regulate FNGPs and AAM. Our findings may help improve the understanding of genetic architecture and pathophysiological mechanisms underlying both osteoporosis and AAM.     

Bivariate Genome-Wide Association Analysis of the Growth and Intake Components of Feed Efficiency

Single nucleotide polymorphisms (SNPs) associated with average daily gain (ADG) and dry matter intake (DMI), two major components of feed efficiency in cattle, were identified in a genome-wide association study (GWAS). Uni- and multi-SNP models were used to describe feed efficiency in a training data set and the results were confirmed in a validation data set. Results from the univariate and bivariate analyses of ADG and DMI, adjusted by the feedlot beef steer maintenance requirements, were compared. The bivariate uni-SNP analysis identified (P-value <0.0001) 11 SNPs, meanwhile the univariate analyses of ADG and DMI identified 8 and 9 SNPs, respectively. Among the six SNPs confirmed in the validation data set, five SNPs were mapped to KDELC2, PHOX2A, and TMEM40. Findings from the uni-SNP models were used to develop highly accurate predictive multi-SNP models in the training data set. Despite the substantially smaller size of the validation data set, the training multi-SNP models had slightly lower predictive ability when applied to the validation data set. Six Gene Ontology molecular functions related to ion transport activity were enriched (P-value <0.001) among the genes associated with the detected SNPs. The findings from this study demonstrate the complementary value of the uni- and multi-SNP models, and univariate and bivariate GWAS analyses. The identified SNPs can be used for genome-enabled improvement of feed efficiency in feedlot beef cattle, and can aid in the design of empirical studies to further confirm the associations.     

Replication of 6 Obesity Genes in a Meta-Analysis of Genome-Wide Association Studies from Diverse Ancestries

Obesity is a major public health problem with a significant genetic component. Multiple DNA polymorphisms/genes have been shown to be strongly associated with obesity, typically in populations of European descent. The aim of this study was to verify the extent to which 6 confirmed obesity genes (FTO, CTNNBL1, ADRB2, LEPR, PPARG and UCP2 genes) could be replicated in 8 different samples (n = 11,161) and to explore whether the same genes contribute to obesity-susceptibility in populations of different ancestries (five Caucasian, one Chinese, one African-American and one Hispanic population). GWAS-based data sets with 1000 G imputed variants were tested for association with obesity phenotypes individually in each population, and subsequently combined in a meta-analysis. Multiple variants at the FTO locus showed significant associations with BMI, fat mass (FM) and percentage of body fat (PBF) in meta-analysis. The strongest association was detected at rs7185735 (P-value = 1.01×10⁻⁷ for BMI, 1.80×10⁻⁶ for FM, and 5.29×10⁻⁴ for PBF). Variants at the CTNNBL1, LEPR and PPARG loci demonstrated nominal association with obesity phenotypes (meta-analysis P-values ranging from 1.15×10⁻³ to 4.94×10⁻²). There was no evidence of association with variants at ADRB2 and UCP2 genes. When stratified by sex and ethnicity, FTO variants showed sex-specific and ethnic-specific effects on obesity traits. Thus, it is likely that FTO has an important role in the sex- and ethnic-specific risk of obesity. Our data confirmed the role of FTO, CTNNBL1, LEPR and PPARG in obesity predisposition. These findings enhanced our knowledge of genetic associations between these genes and obesity-related phenotypes, and provided further justification for pursuing functional studies of these genes in the pathophysiology of obesity. Sex and ethnic differences in genetic susceptibility across populations of diverse ancestries may contribute to a more targeted prevention and customized treatment of obesity.     

TNFR2基因的CA重复多态性在两个独立的白人群体中与肥胖表型的连锁和关联

我们先前通过全基因组扫描发现lp36与体重指数显提示性连锁（LOD=2．09）。肿瘤坏死因子受体2（1NFR2）定位于lp36,是肥胖的一个极好的图位和功能侯选基因。本研究采用数量传递连锁不平衡检验在两个大的独立的白人样本中进行了TNFR2基因与肥胖表型的连锁与关联检验。第一组受试者由来自79个多代家系的1836个个体组成;第二组受试者由来自157个核心家庭的636个个体组成。所检测的肥胖表型包括体重指数、脂肪量和脂肪量百分数。在多代家系中我们发现TNFR2基因变异与BMI显著连锁（P=0．0056）。结果表明,TNFR2基因是影响白人BMI变异的一个数量性状位点。     

Meta-Analysis and Gene Set Analysis of Archived Microarrays Suggest Implication of the Spliceosome in Metastatic and Hypoxic Phenotypes

We propose to make use of the wealth of underused DNA chip data available in public repositories to study the molecular mechanisms behind the adaptation of cancer cells to hypoxic conditions leading to the metastatic phenotype. We have developed new bioinformatics tools and adapted others to identify with maximum sensitivity those genes which are expressed differentially across several experiments. The comparison of two analytical approaches, based on either Over Representation Analysis or Functional Class Scoring, by a meta-analysis-based approach, led to the retrieval of known information about the biological situation – thus validating the model – but also more importantly to the discovery of the previously unknown implication of the spliceosome, the cellular machinery responsible for mRNA splicing, in the development of metastasis.     

Genome-Wide Association Studies and Heritability Estimates of Body Mass Index Related Phenotypes in Bangladeshi Adults

Many health outcomes are influenced by a person''s body mass index, as well as by the trajectory of body mass index through a lifetime. Although previous research has established that body mass index related traits are influenced by genetics, the relationship between these traits and genetics has not been well characterized in people of South Asian ancestry. To begin to characterize this relationship, we analyzed the association between common genetic variation and five phenotypes related to body mass index in a population-based sample of 5,354 Bangladeshi adults. We discovered a significant association between SNV rs347313 (intron of NOS1AP) and change in body mass index in women over two years. In a linear mixed-model, the G allele was associated with an increase of 0.25 kg/m² in body mass index over two years (p-value of 2.3·10⁻⁸). We also estimated the heritability of these phenotypes from our genotype data. We found significant estimates of heritability for all of the body mass index-related phenotypes. Our study evaluated the genetic determinants of body mass index related phenotypes for the first time in South Asians. The results suggest that these phenotypes are heritable and some of this heritability is driven by variation that differs from those previously reported. We also provide evidence that the genetic etiology of body mass index related traits may differ by ancestry, sex, and environment, and consequently that these factors should be considered when assessing the genetic determinants of the risk of body mass index-related disease.     

Linkage and Association Between CA Repeat Polymorphism of the TNFR2 Gene and Obesity Phenotypes in Two Independent Caucasian Populations

Previously, our group has reported a suggestive linkage evidence of 1p36 with body mass index (BMI) (LOD = 2.09). The tumor necrosis factor receptor 2 (TNFR2) at 1p36 is an excellent positional and functional candidate gene for obesity. In this study, we have investigated the linkage and association between the TNFR2 gene and obesity phenotypes in two large independent samples, using the quantitative transmission disequilibrium tests (QTDT). The first group was made up of 1 836 individuals from 79 multi-generation pedigrees. The second group was a randomly ascertained set of 636 individuals from 157 US Caucasian nuclear families. Obesity phenotypes tested include BMI, fat mass, and percentage fat mass (PFM). A significant result (P = 0.0056) was observed for linkage with BMI in the sample of the multigenerational pedigrees. Our data support the TNFR2 gene as a quantitative trait locus (QTL) underlying BMI variation in the Caucasian populations.     

A Genome-Wide Association Study Points out the Causal Implication of SOX9 in the Sex-Reversal Phenotype in XX Pigs

Among farm animals, pigs are known to show XX sex-reversal. In such cases the individuals are genetically female but exhibit a hermaphroditism, or a male phenotype. While the frequency of this congenital disease is quite low (less than 1%), the economic losses are significant for pig breeders. These losses result from sterility, urogenital infections and the carcasses being downgraded because of the risk of boar taint. It has been clearly demonstrated that the SRY gene is not involved in most cases of sex-reversal in pigs, and that autosomal recessive mutations remain to be discovered. A whole-genome scan analysis was performed in the French Large-White population to identify candidate genes: 38 families comprising the two non-affected parents and 1 to 11 sex-reversed full-sib piglets were genotyped with the PorcineSNP60 BeadChip. A Transmission Disequilibrium Test revealed a highly significant candidate region on SSC12 (most significant p-value<4.65.10^-10) containing the SOX9 gene. SOX9, one of the master genes involved in testis differentiation, was sequenced together with one of its main regulatory region Tesco. However, no causal mutations could be identified in either of the two sequenced regions. Further haplotype analyses did not identify a shared homozygous segment between the affected pigs, suggesting either a lack of power due to the SNP properties of the chip, or a second causative locus. Together with information from humans and mice, this study in pigs adds to the field of knowledge, which will lead to characterization of novel molecular mechanisms regulating sexual differentiation and dysregulation in cases of sex reversal.     

Genome-Wide Association Analyses for Fatty Acid Composition in Porcine Muscle and Abdominal Fat Tissues

Fatty acid composition is an important phenotypic trait in pigs as it affects nutritional, technical and sensory quality of pork. Here, we reported a genome-wide association study (GWAS) for fatty acid composition in the longissimus muscle and abdominal fat tissues of 591 White Duroc×Erhualian F₂ animals and in muscle samples of 282 Chinese Sutai pigs. A total of 46 loci surpassing the suggestive significance level were identified on 15 pig chromosomes (SSC) for 12 fatty acids, revealing the complex genetic architecture of fatty acid composition in pigs. Of the 46 loci, 15 on SSC5, 7, 14 and 16 reached the genome-wide significance level. The two most significant SNPs were ss131535508 (P = 2.48×10⁻²⁵) at 41.39 Mb on SSC16 for C20∶0 in abdominal fat and ss478935891 (P = 3.29×10⁻¹³) at 121.31 Mb on SSC14 for muscle C18∶0. A meta-analysis of GWAS identified 4 novel loci and enhanced the association strength at 6 loci compared to those evidenced in a single population, suggesting the presence of common underlying variants. The longissimus muscle and abdominal fat showed consistent association profiles at most of the identified loci and distinct association signals at several loci. All loci have specific effects on fatty acid composition, except for two loci on SSC4 and SSC7 affecting multiple fatness traits. Several promising candidate genes were found in the neighboring regions of the lead SNPs at the genome-wide significant loci, such as SCD for C18∶0 and C16∶1 on SSC14 and ELOVL7 for C20∶0 on SSC16. The findings provide insights into the molecular basis of fatty acid composition in pigs, and would benefit the final identification of the underlying mutations.     

Genome-Wide Survey and Expression Analysis Suggest Diverse Roles of Glutaredoxin Gene Family Members During Development and Response to Various Stimuli in Rice

Glutaredoxins (GRXs) are glutathione-dependent oxidoreductase enzymes involved in a variety of cellular processes. In this study, our analysis revealed the presence of 48 genes encoding GRX proteins in the rice genome. GRX proteins could be classified into four classes, namely CC-, CGFS-, CPYC- and GRL-type, based on phylogenetic analysis. The classification was supported with organization of predicted conserved putative motifs in GRX proteins. We found that expansion of this gene family has occurred largely via whole genome duplication events in a species-specific manner. We explored rice oligonucleotide array data to gain insights into the function of GRX gene family members during various stages of development and in response to environmental stimuli. The comprehensive expression analysis suggested diverse roles of GRX genes during growth and development in rice. Some of the GRX genes were expressed in specific organs/developmental stages only. The expression of many of rice GRX genes was influenced by various phytohormones, abiotic and biotic stress conditions, suggesting an important role of GRX proteins in response to these stimuli. The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provide a new avenue for in-depth characterization of selected genes of importance.     

Association of Proopiomelanocortin Gene Polymorphisms with Obesity in the IRAS Family Study

Proopiomelanocortin (POMC) has been found to be associated with rare Mendelian forms of obesity in children, and, in linkage studies, genomic regions containing the POMC locus have been linked to leptin levels, a predictor of obesity, in white, Mexican‐American, and African‐American families. POMC polymorphisms have not been investigated in detail for association with obesity in the general population. Five single nucleotide polymorphisms (SNPs) (G‐3460C, C17T, G3473A, C3755T, and A7069G) were genotyped on 811 Hispanic individuals in the Insulin Resistance Atherosclerosis Family Study and tested for association with multiple obesity quantitative traits. General and family‐based association analyses for each individual SNP and for haplotypes were performed using the generalized estimating equation and quantitative pedigree disequilibrium test (QPDT), respectively. Modest but consistent associations were observed for SNP C3755T, with p values ranging from 0.011 to 0.045 for association with BMI, waist, visceral adipose tissue, and subcutaneous adipose tissue. G‐3460C, G3473A, and A7069G were also found to be associated with additional obesity measurements (p value 0.025 to 0.04), with comparable levels of evidence observed for linkage disequilibrium between these traits and these SNPs. Results of the haplotype analyses were also consistent with the single SNP analysis, with haplotypes containing C3755T showing the greatest evidence of association (p values ranging 0.004 to 0.048). Monte Carlo simulations (gene dropping) that account for the number of comparisons and the correlation structure indicate that the multivariate significance for these obesity traits with these polymorphisms was p = 0.0091. Collectively, the POMC polymorphisms showed consistent evidence for association with obesity traits in Hispanic Americans across several analytical approaches using SNP and haplotype analysis. These results support the hypothesis that POMC contributes genetically to the development of obesity.     

Meta-analysis Reveals Genome-Wide Significance at 15q13 for Nonsyndromic Clefting of Both the Lip and the Palate,and Functional Analyses Implicate GREM1 As a Plausible Causative Gene

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13×10⁻¹⁴ for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32–1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94–1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47–9.61, P_diff<0.05). While we did not find lip or palate defects in Grem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions.     

Association of Polymorphisms in the Interleukin 6 Receptor Complex with Obesity and Hyperandrogenism

Objective: Interleukin‐6 (IL‐6), is an inflammatory cytokine that may influence the pathogenesis of obesity and hyperandrogenism. IL‐6 exerts its actions through a heterodimeric receptor consisting of two membrane‐bound glycoproteins: an 80‐kDa IL‐6 binding unit (IL6R‐α) and a 130‐kDa IL‐6 signal transducer (gp130). Genetic variability at these loci might contribute to explain the development of obesity and hyperandrogenism. Research Methods and Procedures: We have evaluated the possible association of several polymorphisms in the IL6R‐α and gp130 genes with obesity and/or hyperandrogenism in a case‐control study involving 143 hyperandrogenic patients and 45 healthy women from Spain. Results: A microsatellite CA‐repeat polymorphism in the IL6R‐α locus was associated with obesity. The frequency of the common 149‐bp allele was markedly increased in obese women compared with controls when considering patients and controls as a whole (0.41 vs. 0.29, χ² = 17.085, p < 0.050). On the other hand, the uncommon Arg148 allele of the Gly148Arg polymorphism in the gp130 gene was more frequent in controls compared with hyperandrogenic patients (0.17 vs. 0.08, χ² = 5.605, p = 0.026). Controls carrying Arg148 alleles had lower 11‐deoxycortisol and 17‐hydroxyprogesterone concentrations, a lower response of androstenedione to 1–24 adrenocorticotropin, and an almost significant decrease in free testosterone levels, suggesting that Arg148 alleles in the gp130 gene have a protective effect against androgen excess and adrenal hyperactivity. Discussion: Polymorphisms in the gp130 and IL6R‐α loci influence hyperandrogenism and obesity, respectively. Our present results further suggest that proinflammatory genotypes are involved in the pathogenesis of these common metabolic disorders.     

Study of the Association between Constitutional Exogenous Obesity and Polymorphism of the Apolipoprotein B Gene

An attempt was made to associate the insertion–deletion (Ins/Del) polymorphism of the apolipoprotein B gene (apoB) with obesity and to identify alleles and genotypes predisposing to this disorder. The apoB Ins/Del allele frequencies observed in the Russian population were similar to those in West European populations and significantly differed from frequencies reported for Asian populations. Patients with obesity did not differ from healthy individuals in allele and genotype frequencies regardless of whether total or sex-stratified samples were compared. Estimation of relative risk for individuals with genotype Ins/Ins did not reveal a significant association between obesity and this genotype. Thus, constitutional exogenous obesity did not prove to be associated with the Ins/Del polymorphism of theapoB gene in the Russian population.