Heritability of eleven metabolic phenotypes in Danish and Chinese twins: A cross‐population comparison

Objectives : A twin‐based comparative study on the genetic influences in metabolic endophenotypes in two populations of substantial ethnic, environmental, and cultural differences was performed. Design and Methods : Data on 11 metabolic phenotypes including anthropometric measures, blood glucose, and lipids levels as well as blood pressure were available from 756 pairs of Danish twins (309 monozygotic and 447 dizygotic twin pairs) with a mean age of 38 years (range: 18‐67) and from 325 pairs of Chinese twins (183 monozygotic and 142 dizygotic twin pairs) with a mean age of 40.5 years (range: 18‐69). Twin modeling was performed on full and nested models with the best fitting models selected. Results : Heritability estimates were compared between Danish and Chinese samples to identify differential genetic influences on each of the phenotypes. Except for hip circumference, all other body measures exhibited similar heritability patterns in the two samples with body weight showing only a slight difference. Higher genetic influences were estimated for fasting blood glucose level in Chinese twins, whereas the Danish twins showed more genetic regulation over most lipids phenotypes. Systolic blood pressure was more genetically controlled in Danish than in Chinese twins. Conclusions : Metabolic endophenotypes show disparity in their genetic determinants in populations under distinct environmental conditions.     

Rapid classification of phenotypic mutants of Arabidopsis via metabolite fingerprinting

We evaluated the application of gas chromatography-mass spectrometry metabolic fingerprinting to classify forward genetic mutants with similar phenotypes. Mutations affecting distinct metabolic or signaling pathways can result in common phenotypic traits that are used to identify mutants in genetic screens. Measurement of a broad range of metabolites provides information about the underlying processes affected in such mutants. Metabolite profiles of Arabidopsis (Arabidopsis thaliana) mutants defective in starch metabolism and uncharacterized mutants displaying a starch-excess phenotype were compared. Each genotype displayed a unique fingerprint. Statistical methods grouped the mutants robustly into distinct classes. Determining the genes mutated in three uncharacterized mutants confirmed that those clustering with known mutants were genuinely defective in starch metabolism. A mutant that clustered away from the known mutants was defective in the circadian clock and had a pleiotropic starch-excess phenotype. These results indicate that metabolic fingerprinting is a powerful tool that can rapidly classify forward genetic mutants and streamline the process of gene discovery.     

Genetic and environmental influences on bilateral dental asymmetry in Northwest Indian twins

The role of genetic and environmental factors on dental asymmetry (in maximum crown dimensions) was examined using 58 pairs of twins (23 MZ and 35 DZ) from Chandigarh, India. The t'-test for equality of means by zygosity showed only one variable significantly different among 56: this is ascribable to Type 1 error. Heterogeneity of MZ-DZ total variance was observed in 42.9% of traits of the two types (fluctuating and directional) of bilateral asymmetry. In general, MZ twins showed higher total variance than DZ pairs. MZ twins also showed stronger environmental covariance for a majority of the traits. Dental asymmetry measures thus yielded consistently low genetic variance ratios and indicated predominantly complex environmental determinism. Since fluctuating asymmetry is widely believed to be an environmental stress indicator, this data set allows confirmation of methods for detecting unequal environmental influences on the zygosities which bias estimates of genetic variance and heritability.     

The significance of the homozygous CYP2A6 deletion on nicotine metabolism: a new genotyping method of CYP2A6 using a single PCR-RFLP.

A convenient and specific CYP2A6 genotyping method was developed in this study. This method consisting of a single PCR-RFLP is capable of resolving the genotype into either CYP2A6*1 (wild type), CYP2A6*2, or CYP2A6*3. Among 252 Japanese persons genotyped, 241 were genotyped as the wild type, 1 as an unknown variant, and none as either CYP2A6*2 or CYP2A6*3. A homozygous deletion was found in the 10 remaining subjects. To clarify the metabolic significance of this deletion in the whole human body, urinary cotinine, the principal metabolite of nicotine, was analyzed subsequent to smoking. Cumulated urinary cotinine excretion in the homozygously CYP2A6-deleted individuals was about one-seventh compared to the control group (wild type). This study provides a firm experimental basis for correlating genotypic characterization of CYP2A6 with phenotypic expression of nicotine metabolism.     

Genetic and Environmental Regulation on Longitudinal Change of Metabolic Phenotypes in Danish and Chinese Adult Twins

Objective

The rate of change in metabolic phenotypes can be highly indicative of metabolic disorders and disorder-related modifications. We analyzed data from longitudinal twin studies on multiple metabolic phenotypes in Danish and Chinese twins representing two populations of distinct ethnic, cultural, social-economic backgrounds and geographical environments.

Materials and Methods

The study covered a relatively large sample of 502 pairs of Danish adult twins followed up for a long period of 12 years with a mean age at intake of 38 years (range: 18–65) and a total of 181 Chinese adult twin pairs traced for about 7 years with a mean baseline age of 39.5 years (range: 23–64). The classical twin models were fitted to the longitudinal change in each phenotype (Δphenotype) to estimate the genetic and environmental contributions to the variation in Δphenotype.

Results

Moderate to high contributions by the unique environment were estimated for all phenotypes in both Danish (from 0.51 for low density lipoprotein cholesterol up to 0.72 for triglycerides) and Chinese (from 0.41 for triglycerides up to 0.73 for diastolic blood pressure) twins; low to moderate genetic components were estimated for long-term change in most of the phenotypes in Danish twins except for triglycerides and hip circumference. Compared with Danish twins, the Chinese twins tended to have higher genetic control over the longitudinal changes in lipids (except high density lipoprotein cholesterol) and glucose, higher unique environmental contribution to blood pressure but no genetic contribution to longitudinal change in body mass traits.

Conclusion

Our results emphasize the major contribution of unique environment to the observed intra-individual variation in all metabolic phenotypes in both samples, and meanwhile reveal differential patterns of genetic and common environmental regulation on changes over time in metabolic phenotypes across the two samples.     

Nicotine demethylation in Nicotiana cell suspension cultures: N'-formylnornicotine is not involved

Nicotine or nornicotine enriched with stable isotopes in either the N'-methyl group or the pyrrolidine-N were fed to Nicotiana plumbaginifolia suspension cell cultures that do not form endogenous nicotine. The metabolism of these compounds was investigated by analysing the incorporation of isotope into other alkaloids using gas chromatography-mass spectroscopy (GC-MS). Nicotine metabolism primarily resulted in the accumulation of nornicotine, the N'-demethylation product. In addition, six minor metabolites appeared during the course of nicotine metabolism, four of which were identified as cotinine, myosmine, N'-formylnornicotine and N'-carboethoxynornicotine. While cotinine was formed from [(13)C,(2)H(3)-methyl]nicotine without dilution of label, N'-formylnornicotine was labelled at only about 6% of the level of nicotine and N'-carboethoxynornicotine was unlabelled. Feeding with [1'-(15)N]nornicotine resulted in incorporation without dilution of label into both N'-formylnornicotine and N'-carboethoxynornicotine. This pattern strongly indicates that, while nornicotine and cotinine are derived directly from nicotine, N'-formylnornicotine and N'-carboethoxynornicotine are metabolites of nornicotine. Thus, it is directly demonstrated that N'-formylnornicotine is not an intermediate in nicotine demethylation.     

Acquired obesity is associated with changes in the serum lipidomic profile independent of genetic effects--a monozygotic twin study

Both genetic and environmental factors are involved in the etiology of obesity and the associated lipid disturbances. We determined whether acquired obesity is associated with changes in global serum lipid profiles independent of genetic factors in young adult monozygotic (MZ) twins. 14 healthy MZ pairs discordant for obesity (10 to 25 kg weight difference) and ten weight concordant control pairs aged 24-27 years were identified from a large population-based study. Insulin sensitivity was assessed by the euglycemic clamp technique, and body composition by DEXA (% body fat) and by MRI (subcutaneous and intra-abdominal fat). Global characterization of lipid molecular species in serum was performed by a lipidomics strategy using liquid chromatography coupled to mass spectrometry. Obesity, independent of genetic influences, was primarily related to increases in lysophosphatidylcholines, lipids found in proinflammatory and proatherogenic conditions and to decreases in ether phospholipids, which are known to have antioxidant properties. These lipid changes were associated with insulin resistance, a pathogonomic characteristic of acquired obesity in these young adult twins. Our results show that obesity, already in its early stages and independent of genetic influences, is associated with deleterious alterations in the lipid metabolism known to facilitate atherogenesis, inflammation and insulin resistance.     

Glucocorticoid receptor binding in twin pairs is affected by shared environment but not by shared genes

We set out to determine whether glucocorticoid receptor activity is affected mainly by genetic or environmental factors. The affinity and capacity of the glucocorticoid receptor was measured using dexamethasone binding in whole leukocytes from 53 monozygotic and 48 dizygotic twin pairs. Receptor binding characteristics assayed from twin pairs on the same day were highly correlated within twin pairs irrespective of zygosity. Apparent Kd was negatively correlated with environmental temperature (R²=0.13, P<0.0001) but this did not confound the intra-pair correlation, suggesting a strong familial component independent of zygosity. Receptor binding parameters were not more closely correlated in monozygotic twins than dizygotic twin pairs indicating that there is no major genetic contribution to receptor binding and that environmental influences predominate. The close similarity in binding between twin pairs in adulthood raises the possibility that familial, non-genetic, factors such as shared early life environment may programme the glucocorticoid receptor.     

Biomarkers of exposure to environmental tobacco smoke in infants

Non-invasive biomonitoring of exposure to environmental tobacco smoke (ETS) by means of hair is attractive in children, although systematic evaluation is required in infants. The objective was to compare nicotine and cotinine concentrations in hair and plasma and parentally reported exposure to ETS in a birth cohort of 411 infants. Plasma was collected from 356 six-month-old infants and hair samples were collected from 368 one-year-old infants. Concentrations of nicotine and cotinine were measured by an optimized gas chromatography-mass spectrometry (GC/MS)-based method requiring 4 mg hair or 200 µl plasma. Information was obtained on the number of days with ETS exposure during the first year of life, the smoking habits of the parents, and the number of cigarettes smoked per day in the home. All three parentally reported indices of ETS exposure were significantly associated with the biomarkers, with clear dose-response relationships. There was a significant association between days with ETS exposure and nicotine in hair at relatively low exposure levels (10-99 days per year), whereas the other biomarkers only showed significant increases at higher exposure levels. In conclusion, nicotine in hair appears to be the biomarker most strongly associated with parental reports on exposure to ETS in infants.     

Biomarkers of exposure to environmental tobacco smoke in infants

Non-invasive biomonitoring of exposure to environmental tobacco smoke (ETS) by means of hair is attractive in children, although systematic evaluation is required in infants. The objective was to compare nicotine and cotinine concentrations in hair and plasma and parentally reported exposure to ETS in a birth cohort of 411 infants. Plasma was collected from 356 six-month-old infants and hair samples were collected from 368 one-year-old infants. Concentrations of nicotine and cotinine were measured by an optimized gas chromatography-mass spectrometry (GC/MS)-based method requiring 4 mg hair or 200 µl plasma. Information was obtained on the number of days with ETS exposure during the first year of life, the smoking habits of the parents, and the number of cigarettes smoked per day in the home. All three parentally reported indices of ETS exposure were significantly associated with the biomarkers, with clear dose–response relationships. There was a significant association between days with ETS exposure and nicotine in hair at relatively low exposure levels (10–99 days per year), whereas the other biomarkers only showed significant increases at higher exposure levels. In conclusion, nicotine in hair appears to be the biomarker most strongly associated with parental reports on exposure to ETS in infants.     

The influence of genetic and environmental factors on the etiology of the human umbilical cord: the East Flanders prospective twin survey

The umbilical cord is vulnerable to a number of insults that may alter cord morphology, diminish cord flow, and ultimately compromise fetal nutrition. Thus, an investigation of the underlying mechanisms of the development of cord morphology and possible pathologies associated with it may provide insight regarding fetal growth in the intrauterine environment and have an impact on later development of the child. To our knowledge, this study, which included 11,980 twins, is the first to report the relative contribution of genes and environment in the development of the cord. Umbilical cord length, insertion, knots, twisting, and number of vessels were examined by trained midwives at birth. Means and percentages of cord characteristics by twin zygosity/chorionicity and gender were calculated. ANOVA and chi-square tests were performed to calculate discordance in cord morphology between dizygotic (DZ), monozygotic monochorionic (MZMC), and monozygotic dichorionic (MZDC) twins. Univariate genetic models were fit to the umbilical cord characteristics to investigate the genetic and environmental influences on umbilical cord morphology. Mainly nonshared environmental but also genetic factors influence umbilical cord morphology. In MZMC male and female twins, a peripheral/marginal cord insertion was significantly (P < 0.01) more prevalent compared to MZDC and DZ male and female twins, respectively. In MZMC male twins, clockwise twisting was significantly (P = 0.02) less frequent compared to DZ twins. Environmental and genetic factors influence cord morphology and pathology. Twin members can experience environmental influences that are not shared between them even in that very early stage of in utero life.     

Body odor similarity in noncohabiting twins

There is currently considerable interest in biometric approaches using human odor as a marker of disease or genetic individuality. Body odor is also thought to be used during mate choice to select genetically compatible mates. The idea that body odor reveals information about both genetic identity and genetic similarity is most readily tested by examining odor in twin pairs. However, although this idea can be traced back 130 years to Francis Galton in 1875, most studies using dogs fail to control for shared environmental effects associated with cohabitation. Here we show that odors of identical twins (but not dizygotic twins) can be matched by human sniffers at rates better than chance, even when the twins are living apart. In addition, matching frequencies for identical twin odors were not significantly different from those for duplicate odors from the same individual. These results indicate an important genetic influence on body odor and the potential for developing technologies for human odor printing in relation to underlying genotype.     

Pharmacogenetics of smoking cessation: role of nicotine target and metabolism genes

Many smokers attempt to quit smoking but few are successful in the long term. The heritability of nicotine addiction and smoking relapse have been documented, and research is focused on identifying specific genetic influences on the ability to quit smoking and response to specific medications. Research in genetically modified cell lines and mice has identified nicotine acetylcholine receptor subtypes that mediate the pharmacological and behavioral effects of nicotine sensitivity and withdrawal. Human genetic association studies have identified single nucleotide polymorphisms (SNPs) in genes encoding nicotine acetylcholine receptor subunits and nicotine metabolizing enzymes that influence smoking cessation phenotypes. There is initial promising evidence for a role in smoking cessation for SNPs in the β2 and α5/α3/β4 nAChR subunit genes; however, effects are small and not consistently replicated. There are reproducible and clinically significant associations of genotypic and phenotypic measures of CYP2A6 enzyme activity and nicotine metabolic rate with smoking cessation as well as response to nicotine replacement therapies and bupropion. Prospective clinical trials to identify associations of genetic variants and gene–gene interactions on smoking cessation are needed to generate the evidence base for both medication development and targeted therapy approaches based on genotype.     

Metabolic profiling allows comprehensive phenotyping of genetically or environmentally modified plant systems

Metabolic profiling using gas chromatography-mass spectrometry technologies is a technique whose potential in the field of functional genomics is largely untapped. To demonstrate the general usefulness of this technique, we applied to diverse plant genotypes a recently developed profiling protocol that allows detection of a wide range of hydrophilic metabolites within a single chromatographic run. For this purpose, we chose four independent potato genotypes characterized by modifications in sucrose metabolism. Using data-mining tools, including hierarchical cluster analysis and principle component analysis, we were able to assign clusters to the individual plant systems and to determine relative distances between these clusters. Extraction analysis allowed identification of the most important components of these clusters. Furthermore, correlation analysis revealed close linkages between a broad spectrum of metabolites. In a second, complementary approach, we subjected wild-type potato tissue to environmental manipulations. The metabolic profiles from these experiments were compared with the data sets obtained for the transgenic systems, thus illustrating the potential of metabolic profiling in assessing how a genetic modification can be phenocopied by environmental conditions. In summary, these data demonstrate the use of metabolic profiling in conjunction with data-mining tools as a technique for the comprehensive characterization of a plant genotype.     

Determination of the nicotine metabolites cotinine and trans-3'-hydroxycotinine in biologic fluids of smokers and non-smokers using liquid chromatography-tandem mass spectrometry: biomarkers for tobacco smoke exposure and for phenotyping cytochrome P450 2A6 activity

The nicotine metabolite cotinine is widely used to assess the extent of tobacco use in smokers, and secondhand smoke exposure in non-smokers. The ratio of another nicotine metabolite, trans-3'-hydroxycotinine, to cotinine in biofluids is highly correlated with the rate of nicotine metabolism, which is catalyzed mainly by cytochrome P450 2A6 (CYP2A6). Consequently, this nicotine metabolite ratio is being used to phenotype individuals for CYP2A6 activity and to individualize pharmacotherapies for tobacco addiction. In this paper we describe a highly sensitive liquid chromatography-tandem mass spectrometry method for determination of the nicotine metabolites cotinine and trans-3'-hydroxycotinine in human plasma, urine, and saliva. Lower limits of quantitation range from 0.02 to 0.1ng/mL. The extraction procedure is straightforward and suitable for large-scale studies. The method has been applied to several thousand biofluid samples for pharmacogenetic studies and for studies of exposure to low levels of secondhand smoke. Concentrations of both metabolites in urine of non-smokers with different levels of secondhand smoke exposure are presented.     

Variation in Trans-3′-Hydroxycotinine Glucuronidation Does Not Alter the Nicotine Metabolite Ratio or Nicotine Intake

Background

CYP2A6 metabolizes nicotine to its primary metabolite cotinine and also mediates the metabolism of cotinine to trans-3′-hydroxycotinine (3HC). The ratio of 3HC to cotinine (the “nicotine metabolite ratio”, NMR) is an in vivo marker for the rate of CYP2A6 mediated nicotine metabolism, and total nicotine clearance, and has been associated with differences in numerous smoking behaviors. The clearance of 3HC, which affects the NMR, occurs via renal excretion and metabolism by UGT2B17, and possibly UGT2B10, to 3HC-glucuronide. We investigated whether slower 3HC glucuronidation alters NMR, altering its ability to predict CYP2A6 activity and reducing its clinical utility.

Methods

Plasma NMR, three urinary NMRs, three urinary 3HC glucuronidation phenotypes and total nicotine equivalents were examined in 540 African American smokers. The UGT2B17 gene deletion and UGT2B10*2 were genotyped.

Results

The UGT2B17 gene deletion, but not UGT2B10*2 genotype, was associated with slower 3HC glucuronidation (indicated by three 3HC-glucuronidation phenotypes), indicating its role in this glucuronidation pathway. However, neither lower rates of 3HC glucuronidation, nor the presence of a UGT2B17 and UGT2B10 reduced function allele, altered plasma or urinary NMRs or levels of smoking.

Conclusions

Variation in 3HC glucuronidation activity, including these caused by UGT2B17 gene deletions, did not significantly alter NMR and is therefore unlikely to affect the clinical utility of NMR in smoking behavior and cessation studies. This study demonstrates that NMR is not altered by differences in the rate of 3HC glucuronidation, providing further support that NMR is a reliable indicator of CYP2A6 mediated nicotine metabolism.     

Selected ion monitoring method for determination of nicotine, cotinine and deuterium-labeled analogs: absence of an isotope effect in the clearance of (S)-nicotine-3',3'-d2 in humans

A method for simultaneous determination of nicotine, its metabolite cotinine, and the stable isotope-labeled analogs nicotine-3',3'-d2 and cotinine-4',4'-d2 in human plasma has been developed. The method utilizes capillary column gas chromatography with detection by electron impact mass spectrometry and selected ion monitoring. Sensitivity is adequate for determination of nicotine and nicotine-d2 at concentrations as low as 1 ng ml-1, and cotinine and cotinine-d2 at concentrations as low as 10 ng ml-1 with good precision and accuracy. The method has been used to compare the elimination kinetics of (S)-nicotine-3',3'-d2 with natural nicotine in human subjects. Total clearance of nicotine-3',3'-d2 was virtually identical to the total clearance of natural nicotine, which validates the use of the deuterium-labeled analog in quantitative studies of nicotine metabolic disposition.     

DIURNAL PREFERENCE AND SLEEP QUALITY: SAME GENES? A STUDY OF YOUNG ADULT TWINS

The aims of this study were to examine the genetic and environmental influences on diurnal preference and sleep quality, the association between these phenotypes, the genetic and environmental influences on this association, and the magnitude of overlap between these influences. Using a twin design, data on diurnal preference (measured by the Morningness-Eveningness Questionnaire) and sleep quality (measured by the Pittsburgh Sleep Quality Index) were collected from 420 monozygotic twins, 773 dizygotic twins, and 329 siblings (mode age?=?20 yrs, range?=?18–27 yrs) from a population-based twin registry across the UK. Univariate analyses indicated that dominance genetic influence accounted for 52% and non-shared environment 48% of variance in diurnal preference. For sleep quality, additive genetic influence explained 43% and non-shared environment 57% of the variance. The bivariate analysis indicated a significant association between greater eveningness preference and poorer sleep quality (r?=?.27). There was substantial overlap in the additive genetic influences on both phenotypes (rA?=?.57), and overlap in the dominance genetic influences common to both phenotypes was almost absolute (rD = .99). Overlap in non-shared environment was much smaller (rE?=?.02). Additive genetic influence accounted for 2% of the association, dominance genetic influence accounted for 94%, and non-shared environmental influences accounted for the remaining 4%. The substantial overlap in genetic influence between these phenotypes indicates that similar genes are important for diurnal preference and sleep quality. Therefore, those genes already known to influence one phenotype may be possible candidates to explore with regards to the other phenotype. (Author correspondence: ps701nh@gold.ac.uk)     

Metabolomics characterization of energy metabolism reveals glycogen accumulation in gut-microbiota-lacking mice

Microbiota in the gut are considered an important environmental factor associated with host metabolism and physiology. Although gut microbiota are known to contribute to hepatic lipogenesis and fat storage, little is known about how the condition influences the deposition of glycogen in the liver. To better understand and characterize the host energy metabolism in guts lacking microbiota, we compared the liver metabolome of specific pathogen-free and germ-free mice by gas chromatography-mass spectrometry combined with partial least-squares discriminant analysis. We identified 30 of 52 highly reproducible peaks in chromatograms of liver tissue extracts from the two groups of mice. The two groups showed significant differences in metabolic profile. Changes in liver metabolism involved metabolites such as amino acids, fatty acids, organic acids and carbohydrates. The metabolic profile of germ-free mice suggests that they synthesize glycogen and accumulate it in the liver through gluconeogenesis and glycogenesis. Our findings shed light on a new perspective of the role of gut microbiota in energy metabolism and will be useful to help study probiotics, obesity and metabolic diseases.     

Birth weight and placental proximity in like-sexed twins.

In addition to zygosity, the type of placentation has proven to be an important variable in twin studies. A number of quantitative traits in human twins have been found to be influenced by chorion type. Our study confirms an earlier finding that there is larger within-pair birth weight variability in dichorionic twins with fused placentas than in those with separate placentas. This finding emphasizes the importance of detailed twin placental examinations to help identify traits that may be influenced by prenatal environmental influences.