Statistical challenges in longitudinal microbiome data analysis.

The microbiome is a complex and dynamic community of microorganisms that co-exist interdependently within an ecosystem, and interact with its host or environment. Longitudinal studies can capture temporal variation within the microbiome to gain mechanistic insights into microbial systems; however, current statistical methods are limited due to the complex and inherent features of the data. We have identified three analytical objectives in longitudinal microbial studies: (1) differential abundance over time and between sample groups, demographic factors or clinical variables of interest; (2) clustering of microorganisms evolving concomitantly across time and (3) network modelling to identify temporal relationships between microorganisms. This review explores the strengths and limitations of current methods to fulfill these objectives, compares different methods in simulation and case studies for objectives (1) and (2), and highlights opportunities for further methodological developments. R tutorials are provided to reproduce the analyses conducted in this review.

The newborn metabolome: associations with gestational diabetes, sex, gestation, birth mode, and birth weight.

BACKGROUND: Pathways towards many adult-onset conditions begin early in life, even in utero. Maternal health in pregnancy influences this process, but little is known how it affects neonatal metabolism. We investigated associations between pregnancy and birth factors and cord blood metabolomic profile in a large, population-derived cohort. METHODS: Metabolites were measured using nuclear magnetic resonance in maternal (28 weeks gestation) and cord serum from 912 mother-child pairs in the Barwon Infant Study pre-birth cohort. Associations between maternal (metabolites, age, BMI, smoking), pregnancy (pre-eclampsia, gestational diabetes (GDM)), and birth characteristics (delivery mode, gestational age, weight, infant sex) with 72 cord blood metabolites were examined by linear regression. RESULTS: Delivery mode, sex, gestational age, and birth weight were associated with specific metabolite levels in cord blood, including amino acids, fatty acids, and cholesterols. GDM was associated with higher cord blood levels of acetoacetate and 3-hydroxybutyrate. CONCLUSIONS: Neonatal factors, particularly delivery mode, were associated with many cord blood metabolite differences, including those implicated in later risk of cardiometabolic disease. Associations between GDM and higher offspring ketone levels at birth are consistent with maternal ketosis in diabetic pregnancies. Further work is needed to determine whether these neonatal metabolome differences associate with later health outcomes. IMPACT: Variations in blood metabolomic profile have been linked to health status in adults and children, but corresponding data in neonates are scarce. We report evidence that pregnancy complications, mode of delivery, and offspring characteristics, including sex, are independently associated with a range of circulating metabolites at birth, including ketone bodies, amino acids, cholesterols, and inflammatory markers. Independent of birth weight, exposure to gestational diabetes is associated with higher cord blood ketone bodies and citrate. These findings suggest that pregnancy complications, mode of delivery, gestational age, and measures of growth influence metabolic pathways prior to birth, potentially impacting later health and development.

An integrated metagenomics and metabolomics approach implicates the microbiota-gut-brain axis in the pathogenesis of Huntington's disease.

BACKGROUND: Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder with onset and severity of symptoms influenced by various environmental factors. Recent discoveries have highlighted the importance of the gastrointestinal microbiome in mediating the gut-brain-axis bidirectional communication via circulating factors. Using shotgun sequencing, we investigated the gut microbiome composition in the R6/1 transgenic mouse model of HD from 4 to 12 weeks of age (early adolescent through to adult stages). Targeted metabolomics was also performed on the blood plasma of these mice (n = 9 per group) at 12 weeks of age to investigate potential effects of gut dysbiosis on the plasma metabolome profile. RESULTS: Modelled time profiles of each species, KEGG Orthologs and bacterial genes, revealed heightened volatility in the R6/1 mice, indicating potential early effects of the HD mutation in the gut. In addition to gut dysbiosis in R6/1 mice at 12 weeks of age, gut microbiome function was perturbed. In particular, the butanoate metabolism pathway was elevated, suggesting increased production of the protective SCFA, butyrate, in the gut. No significant alterations were found in the plasma butyrate and propionate levels in the R6/1 mice at 12 weeks of age. The statistical integration of the metagenomics and metabolomics unraveled several Bacteroides species that were negatively correlated with ATP and pipecolic acid in the plasma. CONCLUSIONS: The present study revealed the instability of the HD gut microbiome during the pre-motor symptomatic stage of the disease which may have dire consequences on the host's health. Perturbation of the HD gut microbiome function prior to significant cognitive and motor dysfunction suggest the potential role of the gut in modulating the pathogenesis of HD, potentially via specific altered plasma metabolites which mediate gut-brain signaling.

Sex differences in infant blood metabolite profile in association with weight and adiposity measures.

BACKGROUND: Nuclear magnetic resonance (NMR) metabolic profiling quantifies a large number of metabolites. From adolescence, specific metabolites are influenced by age, sex and body mass index; data on early-life metabolic profiles are limited. We investigated associations between sex, birth weight, weight and adiposity with NMR metabolic profile at age 12 months. METHODS: The plasma NMR metabolic profile was quantified in infants (n = 485) from the Barwon Infant Study. Associations between 74 metabolites and sex, birth weight z-score and 12-month measures (weight z-score, skinfold thickness, weight-for-length z-score) were examined using linear regression models. RESULTS: Several cholesterol and fatty acid measures were higher (0.2-0.3 SD) in girls than in boys; we observed modest sex-specific associations of birth weight z-scores and 12-month sum of skinfold thicknesses with metabolites. The pattern of associations between weight z-score and weight-for-length z-score with metabolites at 12 months was more pronounced in girls, particularly for fatty acid ratios. CONCLUSIONS: We identified sex differences in the infant metabolic profile. Sex-specific patterns observed differ from those reported in older children and adults. We also identified modest cross-sectional associations between anthropometric and adiposity measures and metabolites, some of which were sex specific.

Cross-sectional metabolic profiles of mental health in population-based cohorts of 11- to 12-year-olds and mid-life adults: The Longitudinal Study of Australian Children.

OBJECTIVE: Poorer mental health in adulthood is associated with increased risk of cardiovascular disease and reduced life expectancy. However, little is known of the molecular pathways underpinning this relationship and how early in life adverse metabolite profiles relate to self-reported variation in mental health. We examined cross-sectional associations between mental health and serum metabolites indicative of cardiovascular health, in large Australian population-based cohorts at two stages of the life-course. METHODS: We characterised cross-sectional serum nuclear magnetic resonance metabolite profiles of positively and negatively framed mental health in a large population-based sample of Australian 11- to 12-year-olds (n = 1172; 51% girls) and mid-life adults (n = 1322; mean age 45 years; 87% women). We examined multiple standard self-report mental health scales, spanning psychosocial health, general well-being, life satisfaction, and health-related quality of life. Linear regression was used to investigate the cross-sectional association between mental health and each metabolite (n = 73) in children and adults separately, unadjusted and adjusted for age, sex, socioeconomic position and body mass index. RESULTS: Better child and adult mental health were associated with lower levels of the inflammatory marker glycoprotein acetyls, and a favourable, less atherogenic lipid/lipoprotein profile. Patterns of association in children were generally weaker than in adults. Associations were generally modest and partially attenuated when adjusted for body mass index. CONCLUSIONS: In general, metabolite profiles associated with better child and adult mental health closely aligned with those predictive of better cardiovascular health in adults. Our findings support previous evidence for the likely bidirectional relationship between mental health and cardiovascular disease risk, by extending this evidence base to the molecular level and in children.

Sex and puberty-related differences in metabolomic profiles associated with adiposity measures in youth with obesity.

BACKGROUND: Specific patterns of metabolomic profiles relating to cardiometabolic disease are associated with increased weight in adults. In youth with obesity, metabolomic data are sparse and associations with adiposity measures unknown. OBJECTIVES: Primary, to determine associations between adiposity measures and metabolomic profiles with increased cardiometabolic risks in youth with obesity. Secondary, to stratify associations by sex and puberty. METHODS: Participants were from COBRA (Childhood Overweight BioRepository of Australia; a paediatric cohort with obesity). Adiposity measures (BMI, BMI z-score, %truncal and %whole body fat, waist circumference and waist/height ratio), puberty staging and NMR metabolomic profiles from serum were assessed. Statistics included multivariate analysis (principal component analysis, PCA) and multiple linear regression models with false discovery rate adjustment. RESULTS: 214 participants had metabolomic profiles analyzed, mean age 11.9 years (SD ± 3.1), mean BMI z-score 2.49 (SD ± 0.24), 53% females. Unsupervised PCA identified no separable clusters of individuals. Positive associations included BMI z-score and phenylalanine, total body fat % and lipids in medium HDL, and waist circumference and tyrosine; negative associations included total body fat % and the ratio of docosahexaenoic acid/total fatty acids and histidine. Stratifying by sex and puberty, patterns of associations with BMI z-score in post-pubertal males included positive associations with lipid-, cholesterol- and triglyceride-content in VLDL lipoproteins; total fatty acids; total triglycerides; isoleucine, leucine and glycoprotein acetyls. CONCLUSION: In a paediatric cohort with obesity, increased adiposity measures, especially in post-pubertal males, were associated with distinct patterns in metabolomic profiles.

The Australian Aboriginal Birth Cohort study: socio-economic status at birth and cardiovascular risk factors to 25 years of age.

OBJECTIVES: To determine whether socio-economic status at birth is associated with differences in risk factors for cardiovascular disease - body mass index (BMI), blood pressure, blood lipid levels - during the first 25 years of life. DESIGN: Analysis of prospectively collected data. SETTING, PARTICIPANTS: 570 of 686 children born to Aboriginal mothers at the Royal Darwin Hospital during 1987-1990 and recruited for the Aboriginal Birth Cohort Study in the Northern Territory. Participants resided in 46 urban and remote communities across the NT. The analysed data were collected at three follow-ups: Wave 2 in 1998-2001 (570 participants; mean age, 11 years), Wave 3 in 2006-2008 (442 participants; mean age, 18 years), and Wave 4 in 2014-2016 (423 participants; mean age, 25 years). MAIN OUTCOME MEASURES: Cardiovascular disease risk factors by study wave and three socio-economic measures at the time of birth: area-level Indigenous Relative Socioeconomic Outcomes (IRSEO) index score and location (urban, remote) of residence, and parity of mother. RESULTS: Area-level IRSEO of residence at birth influenced BMI (P < 0.001), systolic blood pressure (P = 0.024), LDL-cholesterol (P = 0.010), and HDL-cholesterol levels (P < 0.001). Remoteness of residence at birth influenced BMI (P < 0.001), HDL-cholesterol (P < 0.001), and triglyceride levels (P = 0.043). Mother's parity at birth influenced BMI (P = 0.039). CONCLUSIONS: Our longitudinal life course analyses indicate that area-level socio-economic factors at birth influence the prevalence of major cardiovascular disease risk factors among Indigenous Australians during childhood and early adulthood.

Brachial-cuff excess pressure is associated with carotid intima-media thickness among Australian children: a cross-sectional population study.

Reservoir pressure parameters (i.e., reservoir pressure [RP] and excess pressure [XSP]) independently predict cardiovascular events in adults, but this has not been investigated in children. This study aimed to determine (1) the association of reservoir pressure parameters with carotid intima-media thickness (carotid IMT), a preclinical vascular phenotype, and (2) whether a multivariable regression model with or without reservoir pressure parameters fits better for estimating carotid IMT in children. Study participants were 11-12-year-old children (n = 1231, 50% male) from the Child Health CheckPoint study, a cross-sectional substudy of the population-based Longitudinal Study of Australian Children. RP and XSP were obtained using brachial-cuff oscillometry (SphygmoCor XCEL, AtCor, Sydney). Carotid IMT was quantified by vascular ultrasonography. XSP was associated with carotid IMT after adjusting for confounders including age, sex, BMI z-score, heart rate, pubertal stage, moderate-to-vigorous physical activity, and mean arterial pressure (ß = 0.93 µm, 95% CI 0.30-1.56 for XSP peak and ß = 0.04 µm, 95% CI 0.01-0.08 for XSP integral). The results of the likelihood ratio test indicated a trend that the model with XSP and the above confounders fit better than a similar model without XSP for estimating carotid IMT. Our findings indicate that brachial-cuff device-measured XSP is associated with carotid IMT independent of conventional cardiovascular risk factors, including standard BP. This implies that a clinically convenient cuff approach could provide meaningful information for the early assessment of cardiovascular risk among children.

Plasma metabolomic profiles associated with infant food allergy with further consideration of other early life factors.

BACKGROUND: Fatty acids have been implicated in early life immune development. Food allergy provides a clear phenotype of early allergic disease. Fish oil and vitamin D have immune-modulating properties. We aimed to identify the metabolomic profile of (i) infant food allergy and (ii) factors linked to food allergy in past studies such as fish oil supplementation and serum 25OHD3 levels in early life. METHODS: NMR was used to quantify 73 metabolites in plasma of 1 year old infants from the Barwon Infant Study (n=485). Logistic regression models were used to examine associations between infant metabolome and food allergy in infants. Linear regression models were used to describe associations between maternal fish oil supplementation and 25OHD3 levels with infant metabolites. RESULTS: A higher linoleic acid: total fatty acid (FA) ratio and phenylalanine level were associated with higher odds of food allergy. Antenatal fish oil supplementation was positively associated with docosahexaenoic acid (DHA) and omega-3 related metabolite levels. Postnatal 25OHD3 levels at 1 year of age were positively associated with several FA measures and creatinine and inversely with the saturated FA: total FA ratio. Only the postnatal 25OHD3 patterns persisted after adjustment for multiple comparisons. CONCLUSIONS: Infants with food allergy had altered fatty acid profiles at one year. Fish oil supplementation in pregnancy was associated with higher DHA and omega-3 related metabolites at 1 year of age. Associations were modest and the most robustly altered metabolomic profiles were with postnatal 25OHD3 levels.

Association of brachial-cuff excess pressure with carotid intima-media thickness in Australian adults: a cross-sectional study.

OBJECTIVE: Reservoir pressure parameters [e.g. reservoir pressure (RP) and excess pressure (XSP)] measured using tonometry predict cardiovascular events beyond conventional risk factors. However, the operator dependency of tonometry impedes widespread use. An operator-independent cuff-based device can reasonably estimate the intra-aortic RP and XSP from brachial volumetric waveforms, but whether these estimates are clinically relevant to preclinical phenotypes of cardiovascular risk has not been investigated. METHODS: The RP and XSP were derived from brachial volumetric waveforms measured using cuff oscillometry (SphygmoCor XCEL) in 1691 mid-life adults from the CheckPoint study (a population-based cross-sectional study nested in the Longitudinal Study of Australian Children). Carotid intima--media thickness (carotid IMT, n = 1447) and carotid--femoral pulse wave velocity (PWV, n = 1632) were measured as preclinical phenotypes of cardiovascular risk. Confounders were conventional risk factors that were correlated with both exposures and outcomes or considered as physiologically important. RESULTS: There was a modest association between XSP and carotid IMT (ß = 0.76 µm, 95% CI, 0.25-1.26 partial R = 0.8%) after adjusting for age, sex, BMI, heart rate, smoking, diabetes, high-density lipoprotein cholesterol and mean arterial pressure. Neither RP nor XSP were associated with PWV in the similarly adjusted models (ß = -0.47 cm/s, 95% CI, -1.15 to 0.20, partial R = 0.2% for RP, and ß = 0.04 cm/s, 95% CI, -0.59 to 0.67, partial R = 0.01% for XSP). CONCLUSION: Cuff-based XSP associates with carotid IMT independent of conventional risk factors, including traditional BP, but the association was weak, indicating that further investigation is warranted to understand the clinical significance of reservoir pressure parameters.

A Cross-Cohort Study Examining the Associations of Metabolomic Profile and Subclinical Atherosclerosis in Children and Their Parents: The Child Health CheckPoint Study and Avon Longitudinal Study of Parents and Children.

Background High-throughput nuclear magnetic resonance profiling of circulating metabolites is suggested as an adjunct for cardiovascular risk evaluation. The relationship between metabolites and subclinical atherosclerosis remains unclear, particularly among children. Therefore, we examined the associations of metabolites with carotid intima-media thickness ( cIMT ) and arterial pulse wave velocity ( PWV ). Methods and Results Data from two independent population-based studies was examined; (1) cross-sectional associations with cIMT and PWV in 1178 children (age 11-12 years, 51% female) and 1316 parents (mean age 45 years, 87% female) from the CheckPoint study (Australia); and (2) longitudinal associations in 4249 children (metabolites at 7-8 years, PWV at 10-11 years, 52% female), and cross-sectional associations in 4171 of their mothers (mean age 48 years, cIMT data) from ALSPAC (The Avon Longitudinal Study of Parents and Children; UK ). Metabolites were measured by the same nuclear magnetic resonance platform in both studies, comprising of 69 biomarkers. Biophysical assessments included body mass index, blood pressure, cIMT and PWV . In linear regression analyses adjusted for age, sex, body mass index, and blood pressure, there was no evidence of metabolite associations in either children or adults for cIMT at a 10% false discovery threshold. In CheckPoint adults, glucose was positively, and some high-density lipoprotein-cholesterol derived measures and amino acids (glutamine, histidine, tyrosine) inversely associated with PWV. Conclusions These data suggest that in children circulating metabolites have no consistent association with cIMT and PWV once adjusted for body mass index and blood pressure. In their middle-aged parents, some evidence of metabolite associations with PWV were identified that warrant further investigation.

Metabolomics: population epidemiology and concordance in Australian children aged 11-12 years and their parents.

OBJECTIVES: Nuclear magnetic resonance (NMR) metabolomics is high throughput and cost-effective, with the potential to improve the understanding of disease and risk. We examine the circulating metabolic profile by quantitative NMR metabolomics of a sample of Australian 11-12 year olds children and their parents, describe differences by age and sex, and explore the correlation of metabolites in parent-child dyads. DESIGN: The population-based cross-sectional Child Health CheckPoint study nested within the Longitudinal Study of Australian Children. SETTING: Blood samples collected from CheckPoint participants at assessment centres in seven Australian cities and eight regional towns; February 2015-March 2016. PARTICIPANTS: 1180 children and 1325 parents provided a blood sample and had metabolomics data available. This included 1133 parent-child dyads (518 mother-daughter, 469 mother-son, 68 father-daughter and 78 father-son). OUTCOME MEASURES: 228 metabolic measures were obtained for each participant. We focused on 74 biomarkers including amino acid species, lipoprotein subclass measures, lipids, fatty acids, measures related to fatty acid saturation, and composite markers of inflammation and energy homeostasis. RESULTS: We identified differences in the concentration of specific metabolites between childhood and adulthood and in metabolic profiles in children and adults by sex. In general, metabolite concentrations were higher in adults than children and sex differences were larger in adults than in children. Positive correlations were observed for the majority of metabolites including isoleucine (CC 0.33, 95% CI 0.27 to 0.38), total cholesterol (CC 0.30, 95% CI 0.24 to 0.35) and omega 6 fatty acids (CC 0.28, 95% CI 0.23 to 0.34) in parent-child comparisons. CONCLUSIONS: We describe the serum metabolite profiles from mid-childhood and adulthood in a population-based sample, together with a parent-child concordance. Differences in profiles by age and sex were observed. These data will be informative for investigation of the childhood origins of adult non-communicable diseases and for comparative studies in other populations.