Alexis E. Duncan

A core gut microbiome in obese and lean twins

The human distal gut harbours a vast ensemble of microbes (the microbiota) that provide important metabolic capabilities, including the ability to extract energy from otherwise indigestible dietary polysaccharides. Studies of a few unrelated, healthy adults have revealed substantial diversity in their gut communities, as measured by sequencing 16S rRNA genes, yet how this diversity relates to function and to the rest of the genes in the collective genomes of the microbiota (the gut microbiome) remains obscure. Studies of lean and obese mice suggest that the gut microbiota affects energy balance by influencing the efficiency of calorie harvest from the diet, and how this harvested energy is used and stored. Here we characterize the faecal microbial communities of adult female monozygotic and dizygotic twin pairs concordant for leanness or obesity, and their mothers, to address how host genotype, environmental exposure and host adiposity influence the gut microbiome. Analysis of 154 individuals yielded 9,920 near full-length and 1,937,461 partial bacterial 16S rRNA sequences, plus 2.14 gigabases from their microbiomes. The results reveal that the human gut microbiome is shared among family members, but that each person’s gut microbial community varies in the specific bacterial lineages present, with a comparable degree of co-variation between adult monozygotic and dizygotic twin pairs. However, there was a wide array of shared microbial genes among sampled individuals, comprising an extensive, identifiable ‘core microbiome’ at the gene, rather than at the organismal lineage, level. Obesity is associated with phylum-level changes in the microbiota, reduced bacterial diversity and altered representation of bacterial genes and metabolic pathways. These results demonstrate that a diversity of organismal assemblages can nonetheless yield a core microbiome at a functional level, and that deviations from this core are associated with different physiological states (obese compared with lean).

Gut microbiota from twins discordant for obesity modulate metabolism in mice.

Introduction Establishing whether specific structural and functional configurations of a human gut microbiota are causally related to a given physiologic or disease phenotype is challenging. Twins discordant for obesity provide an opportunity to examine interrelations between obesity and its associated metabolic disorders, diet, and the gut microbiota. Transplanting the intact uncultured or cultured human fecal microbiota from each member of a discordant twin pair into separate groups of recipient germfree mice permits the donors’ communities to be replicated, differences between their properties to be identified, the impact of these differences on body composition and metabolic phenotypes to be discerned, and the effects of diet-by-microbiota interactions to be analyzed. In addition, cohousing coprophagic mice harboring transplanted microbiota from discordant pairs provides an opportunity to determine which bacterial taxa invade the gut communities of cage mates, how invasion correlates with host phenotypes, and how invasion and microbial niche are affected by human diets. Cohousing Ln and Ob mice prevents increased adiposity in Ob cage mates (Ob). (A) Adiposity change after 10 days of cohousing. *P < 0.05 versus Ob controls (Student’s t test). (B) Bacteroidales from Ln microbiota invade Ob microbiota. Columns show individual mice. Methods Separate groups of germfree mice were colonized with uncultured fecal microbiota from each member of four twin pairs discordant for obesity or with culture collections from an obese (Ob) or lean (Ln) co-twin. Animals were fed a mouse chow low in fat and rich in plant polysaccharides, or one of two diets reflecting the upper or lower tertiles of consumption of saturated fats and fruits and vegetables based on the U.S. National Health and Nutrition Examination Survey (NHANES). Ln or Ob mice were cohoused 5 days after colonization. Body composition changes were defined by quantitative magnetic resonance. Microbiota or microbiome structure, gene expression, and metabolism were assayed by 16S ribosomal RNA profiling, whole-community shotgun sequencing, RNA-sequencing, and mass spectrometry. Host gene expression and metabolism were also characterized. Results and Discussion The intact uncultured and culturable bacterial component of Ob co-twins’ fecal microbiota conveyed significantly greater increases in body mass and adiposity than those of Ln communities. Differences in body composition were correlated with differences in fermentation of short-chain fatty acids (increased in Ln), metabolism of branched-chain amino acids (increased in Ob), and microbial transformation of bile acid species (increased in Ln and correlated with down-regulation of host farnesoid X receptor signaling). Cohousing Ln and Ob mice prevented development of increased adiposity and body mass in Ob cage mates and transformed their microbiota’s metabolic profile to a leanlike state. Transformation correlated with invasion of members of Bacteroidales from Ln into Ob microbiota. Invasion and phenotypic rescue were diet-dependent and occurred with the diet representing the lower tertile of U.S. consumption of saturated fats, and upper tertile of fruits and vegetables, but not with the diet representing the upper tertile of saturated fats, and lower tertile of fruit and vegetable consumption. These results reveal that transmissible and modifiable interactions between diet and microbiota influence host biology. Transforming Fat to Thin How much does the microbiota influence the hosts phenotype? Ridaura et al. (1241214 ; see the Perspective by Walker and Parkhill) obtained uncultured fecal microbiota from twin pairs discordant for body mass and transplanted them into adult germ-free mice. It was discovered that adiposity is transmissible from human to mouse and that it was associated with changes in serum levels of branched-chain amino acids. Moreover, obese-phenotype mice were invaded by members of the Bacteroidales from the lean mice, but, happily, the lean animals resisted invasion by the obese microbiota. Mice carrying gut bacteria from lean humans protect their cage mates from the effects of gut bacteria from fat humans. [Also see Perspective by Walker and Parkhill] The role of specific gut microbes in shaping body composition remains unclear. We transplanted fecal microbiota from adult female twin pairs discordant for obesity into germ-free mice fed low-fat mouse chow, as well as diets representing different levels of saturated fat and fruit and vegetable consumption typical of the U.S. diet. Increased total body and fat mass, as well as obesity-associated metabolic phenotypes, were transmissible with uncultured fecal communities and with their corresponding fecal bacterial culture collections. Cohousing mice harboring an obese twin’s microbiota (Ob) with mice containing the lean co-twin’s microbiota (Ln) prevented the development of increased body mass and obesity-associated metabolic phenotypes in Ob cage mates. Rescue correlated with invasion of specific members of Bacteroidetes from the Ln microbiota into Ob microbiota and was diet-dependent. These findings reveal transmissible, rapid, and modifiable effects of diet-by-microbiota interactions.

The Impact of a Consortium of Fermented Milk Strains on the Gut Microbiome of Gnotobiotic Mice and Monozygotic Twins

Metagenomic analyses of gnotobiotic mice and monozygotic twins reveal the effects of eating a popular fermented milk product on their microbiomes. A Yogurt a Day… We all enjoy a tasty yogurt and believe that the bacterial species contained in this type of fermented milk product will keep us healthy. But how much influence do the microbes in these products have on our gut microbiomes and consequently our health, and are these effects generalizable to different human populations consuming different diets? These questions are of concern to regulatory agencies who are increasing pressure on manufacturers to validate the health claims of various foods, including yogurts. McNulty and his colleagues, in an exciting new study, describe a way to evaluate their effects on the human gut microbiome. First, they studied the effects of consuming a popular yogurt on the gut microbiomes of seven healthy adult female identical twin pairs. The bacterial and gene composition, as well as the gene expression patterns, of their gut microbial communities were analyzed before, during, and after consumption of the yogurt. These results were compared to those obtained in gnotobiotic mice that were first reared under conditions where the only microbes they harbored were 15 prominent, sequenced human gut bacterial symbionts, after which time they were exposed to the same 5 bacterial strains as those contained in the yogurt. McNulty and colleagues found during repeated sampling of the gut microbiomes of the twins over a 4-month period that the species and gene content of their gut microbial communities remained stable and were not appreciably perturbed by consuming the yogurt. After exposure of the humanized mice to the five bacterial strains in the fermented milk product, the researchers showed that the mice did not exhibit marked changes in the proportional representation of their human symbiotic bacterial species or genes, mirroring the results seen in the twins. However, analysis of gut bacterial gene expression profiles and of urinary metabolites in these mice disclosed that introducing the fermented milk product strains resulted in marked changes in a number of metabolic pathways, most prominently those related to carbohydrate processing. These latter findings helped direct follow-up studies of the twins’ gut samples where they found similar changes in metabolism as those observed in mice. These findings show that mice containing a sequenced model human gut microbiome can serve as part of a preclinical discovery pipeline designed to identify the effects of existing or new bacterial species with purported health benefits on the properties of the human gut microbiome. Although it remains unclear whether eating a yogurt a day will keep the doctor away, the study by McNulty and his colleagues paves the way for future work to analyze in more detail the direct effects of consuming foods containing bacterial species with potential health benefits on the gut microbiomes of various human populations. Understanding how the human gut microbiota and host are affected by probiotic bacterial strains requires carefully controlled studies in humans and in mouse models of the gut ecosystem where potentially confounding variables that are difficult to control in humans can be constrained. Therefore, we characterized the fecal microbiomes and metatranscriptomes of adult female monozygotic twin pairs through repeated sampling 4 weeks before, 7 weeks during, and 4 weeks after consumption of a commercially available fermented milk product (FMP) containing a consortium of Bifidobacterium animalis subsp. lactis, two strains of Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis subsp. cremoris, and Streptococcus thermophilus. In addition, gnotobiotic mice harboring a 15-species model human gut microbiota whose genomes contain 58,399 known or predicted protein-coding genes were studied before and after gavage with all five sequenced FMP strains. No significant changes in bacterial species composition or in the proportional representation of genes encoding known enzymes were observed in the feces of humans consuming the FMP. Only minimal changes in microbiota configuration were noted in mice after single or repeated gavage with the FMP consortium. However, RNA-Seq analysis of fecal samples and follow-up mass spectrometry of urinary metabolites disclosed that introducing the FMP strains into mice results in significant changes in expression of microbiome-encoded enzymes involved in numerous metabolic pathways, most prominently those related to carbohydrate metabolism. B. animalis subsp. lactis, the dominant persistent member of the FMP consortium in gnotobiotic mice, up-regulates a locus in vivo that is involved in the catabolism of xylooligosaccharides, a class of glycans widely distributed in fruits, vegetables, and other foods, underscoring the importance of these sugars to this bacterial species. The human fecal metatranscriptome exhibited significant changes, confined to the period of FMP consumption, that mirror changes in gnotobiotic mice, including those related to plant polysaccharide metabolism. These experiments illustrate a translational research pipeline for characterizing the effects of FMPs on the human gut microbiome.

Common genetic and environmental contributions to post-traumatic stress disorder and alcohol dependence in young women.

BACKGROUND The few genetically informative studies to examine post-traumatic stress disorder (PTSD) and alcohol dependence (AD), all of which are based on a male veteran sample, suggest that the co-morbidity between PTSD and AD may be attributable in part to overlapping genetic influences, but this issue has yet to be addressed in females.MethodData were derived from an all-female twin sample (n=3768) ranging in age from 18 to 29 years. A trivariate genetic model that included trauma exposure as a separate phenotype was fitted to estimate genetic and environmental contributions to PTSD and the degree to which they overlap with those that contribute to AD, after accounting for potential confounding effects of heritable influences on trauma exposure. RESULTS Additive genetic influences (A) accounted for 72% of the variance in PTSD; individual-specific environmental (E) factors accounted for the remainder. An AE model also provided the best fit for AD, for which heritability was estimated to be 71%. The genetic correlation between PTSD and AD was 0.54. CONCLUSIONS The heritability estimate for PTSD in our sample is higher than estimates reported in earlier studies based almost exclusively on an all-male sample in which combat exposure was the precipitating traumatic event. However, our findings are consistent with the absence of evidence for shared environmental influences on PTSD and, most importantly, the substantial overlap in genetic influences on PTSD and AD reported in these investigations. Additional research addressing potential distinctions by gender in the relative contributions of genetic and environmental influences on PTSD is merited.

The effects of maternal smoking during pregnancy on offspring outcomes

OBJECTIVE To evaluate the possible association between maternal smoking during pregnancy and offspring outcomes of birth weight, pre-term birth, remediation, low scholastic achievement, regular smoking, attention deficit hyperactivity disorder and conduct problems while controlling for similar behaviors in parents. METHODS Using telephone interviews, data were collected, in 2001 and 2004, as a part of two United States offspring-of-twins projects. Fathers, who were twins participating in the Vietnam Era Twin Registry, their female spouse and their offspring were interviewed - information on 1,342 unique pregnancies in mothers with a history of regular smoking was utilized for these analyses. The association between maternal smoking during pregnancy and birth weight, pre-term birth, remediation, low scholastic achievement, regular smoking, attention deficit hyperactivity disorder and conduct disorder while controlling for similar behaviors in parents, was examined using regression. RESULTS Maternal smoking during pregnancy was associated with decreased birth weight, low scholastic achievement, regular smoking and attention deficit hyperactivity disorder. However, the association between maternal smoking during pregnancy and offspring attention deficit hyperactivity disorder was explained by maternal attention deficit hyperactivity disorder. Maternal smoking during pregnancy was also associated with earlier age of offspring initiation of smoking and onset of regular smoking. CONCLUSIONS Maternal smoking during pregnancy may influence certain offspring outcomes via mechanisms that are independent from genetic risk attributable to comorbid conditions. Assisting expecting mothers with their smoking cessation efforts will likely provide widespread health benefits to both mother and offspring.

The association between cannabis abuse and dependence and childhood physical and sexual abuse: evidence from an offspring of twins design

AIM This study examines the association between childhood physical abuse (CPA) and sexual abuse (CSA) and the development of cannabis abuse and dependence among adolescents and young adults while controlling for genetic and environmental risk factors. DESIGN To control for familial risk differences related to paternal drug dependence that might confound the relationship between CSA and CPA and cannabis abuse/dependence, we created four groups based on fathers and uncles substance use dependence (SUD) status reflecting different degrees of genetic and environmental risks to offspring: (i) high genetic, high environmental risk; (ii) high genetic, low environmental risk; (iii) medium genetic, low environmental risk; and (iv) low genetic, low environmental risk. PARTICIPANTS Adolescent and young adult offspring of monozygotic and dizygotic US military veteran twin fathers (n = 819). MEASUREMENTS Data on CPA and CSA, DSM-IV offspring cannabis abuse/dependence, other SUD and psychopathology and maternal and paternal SUD and psychopathology were collected via semi-structured telephone interview. FINDINGS Twenty-three per cent of the offspring sample met life-time criteria for cannabis abuse/dependence and 8.55% and 12.82% reported CSA and CPA, respectively. Offspring exposed to CSA, but not CPA, were at significantly greater risk of developing cannabis abuse/dependence compared to those who had not experienced CSA (hazard ratio = 2.16; 95% confidence interval = 1.48-3.16) after controlling for genetic and familial environmental risk and offspring gender, alcohol abuse and dependence and conduct disorder. CONCLUSIONS These results indicate that there are effects of CSA on development of cannabis abuse/dependence in addition to the genetic and familial environmental risk imparted by having a drug-dependent father.

Associations of alcohol, nicotine, cannabis, and drug use/dependence with educational attainment: evidence from cotwin-control analyses

BACKGROUND Although substance use is associated with reduced educational attainment, this association may be owing to common risk factors such as socioeconomic disadvantage. We tested whether alcohol, nicotine, and illicit drug use and dependence were associated with lifetime educational attainment after controlling for familial background characteristics. METHODS Data were from a 1987 questionnaire and a 1992 telephone diagnostic interview of 6,242 male twins (n = 3,121 pairs; mean age = 41.9 years in 1992) who served in the U.S. military during the Vietnam era and therefore, were eligible for educational benefits after military service. Reduced educational attainment (<16 years) was examined in twin pairs discordant for substance use history. Substance use and dependence risk factors assessed were early alcohol and cannabis use, daily nicotine use, lifetime cannabis use, and alcohol, nicotine, cannabis, and any illicit drug dependence. RESULTS Three significant differences were observed between at-risk twins and their cotwins: Compared to their low-risk cotwins, likelihood of completing <16 years of education was significantly increased for the following: (i) twins who used alcohol before age 18 (adjusted OR = 1.44; 95% CI: 1.02 to 2.05), (ii) twins with a lifetime alcohol dependence diagnosis (adjusted OR = 1.76; 95% CI: 1.27 to 2.44), and (iii) twins who had used nicotine daily for 30 or more days (adjusted OR = 2.54, 95% CI: 1.55 to 4.17). However, no differences in education were observed among twin pairs discordant for cannabis initiation, early cannabis use, or cannabis, nicotine, or any illicit drug dependence. CONCLUSIONS Even in a veteran population with access to military educational benefits, early alcohol use, alcohol dependence, and daily nicotine use remained significantly associated with years of education after controlling for shared familial contributions to educational attainment. The association between other substances and educational attainment was explained by familial factors common to these substance use phenotypes and adult educational attainment.

Genome-wide association study of height and body mass index in Australian twin families

Human height and body mass index are influenced by a large number of genes, each with small effects, along with environment. To identify common genetic variants associated with these traits, we performed genome-wide association studies in 11,536 individuals composed of Australian twins, family members, and unrelated individuals at approximately 550,000 genotyped SNPs. We identified a single genome-wide significant variant for height (P value=1.06x10(-9)) located in HHIP, a well-replicated height-associated gene. Suggestive levels of association were found for other known genes associated with height (P values<1x10(-6)): ADAMTSL3, EFEMP1, GPR126, and HMGA2; and BMI (P values<1x10(-4)): FTO and MC4R. Together, these variants explain less than 2% of total phenotypic variation for height and 0.5% for BMI.

Clustering of eating disorder symptoms in a general population female twin sample: A latent class analysis

BACKGROUND Previous studies have reported that the current DSM-IV eating disorder (ED) criteria do not adequately describe ED symptomatology. The objective of the current study was to examine the clustering of ED symptoms in a general population sample using latent class analysis (LCA). METHOD ED symptoms from 3723 female young adult twins (mean age 22) were analyzed using LCA, and resulting classes were compared on external validators reflecting ED and other co-morbid psychiatric diagnoses, substance use disorders (SUDs), and suicidality. RESULTS The optimal solution consisted of five latent classes characterized as: (1) Unaffected; (2) Low Weight Gain; (3) Weight Concerned; (4) Dieters; and (5) ED. Members of the ED class had significantly higher prevalence of co-morbid psychiatric disorders, SUDs, and suicidality than the Unaffected and Low Weight Gain classes, and elevated rates of suicidality and major depression compared to the Weight Concerned and Dieter classes, which differed from each other primarily in terms of current body mass index (BMI). Dieter class members were more likely to be overweight and obese and less likely to be underweight than Weight Concerned class members. The majority of women with an ED diagnosis were assigned to the ED class, and few differences were found between ED class members with and without an ED diagnosis. CONCLUSIONS The results add to the evidence that many women with significant ED psychopathology are not being identified by the DSM-IV ED categories.

Childhood sexual abuse and early substance use in adolescent girls: the role of familial influences

AIM To assess the extent to which the association between childhood sexual abuse (CSA) and early use of alcohol, cigarettes and cannabis in adolescent girls is mediated by risk factors that tend to cluster in families where CSA occurs. DESIGN An abridged version of the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) was administered by telephone. PARTICIPANTS A total of 3761 female twins aged 18-29 (14.6% African American, 85.4% European American). MEASUREMENTS CSA experiences and history of substance use were queried in the SSAGA-based interviews. FINDINGS After controlling for familial influences on early substance use by including co-twin early use status in models, separate Cox proportional hazards regression analyses predicting onset of alcohol, cigarette and cannabis use revealed a significant association with CSA. The effect was observed to age 19 years for cigarettes and to age 21 years for cannabis, but was limited to age 14 years or younger for alcohol, with the most pronounced risk before age 10 [hazard ratio (HR) = 4.59; confidence interval (CI): 1.96-10.74]. CSA-associated risk for initiation of cigarette and cannabis use was also highest in the youngest age range, but the decline with age was much more gradual and the hazard ratios significantly lower (HR: 1.70; CI: 1.13-2.56 for cigarettes and HR: 2.34, CI: 1.57-3.48 for cannabis). CONCLUSIONS Childhood sexual abuse history is a distinct risk factor for use of cigarettes and cannabis, and a very strong predictor of early age at first drink.