Micronutrients

START has been granted ethical approval locally from the Research Ethics Board, Hamilton Health Sciences/McMaster Health Sciences (REB#: 10-640) and in India, Institutional Ethics Review Board Reference #: 114/2010). In both countries, pregnant mothers are recruited during their antenatal visits (1^st or 2^nd trimester) to their primary care practitioner or obstetrician. The study is described by the study personnel to the pregnant mothers and consent for participation is obtained. Information concerning medical and pregnancy history, health status, health behaviors, and socioeconomic status is obtained by questionnaires. Anthropometric measurements (height, weight, skinfold thickness), blood pressure, urine sample, and a fasting blood sample for glucose, insulin, micronutrients (i.e. vitamin B12, RBC folate, plasma homocysteine, methylmalonic acid MMA), lipids and a buffy coat for future DNA extraction will be collected, and processed using a standardized protocol at 24-28 weeks of gestation. Mothers who are not known to have diabetes will undergo a 75 oral glucose tolerance test between 24-28 weeks gestation. The results of an ultrasound performed between 18-24 weeks to assess for congenital anomalies and for precise determination of gestational age will be collected from each pregnant mother. At the time of delivery, details of the delivery, birth outcomes for the mother and baby will be collected, and a cord blood sample for DNA, glucose, insulin, lipids and additional aliquots for future analysis of adiponectin, and leptin will be taken from each baby. The placenta will be weighed, and where possible a biopsy of the placenta will be collected and stored in RNAlater for future analysis of RNA and methylation patterns. In addition, the infant’s anthropometry including birth weight, triceps and sub-scapular skin fold thickness, length, abdominal, head, and arm circumference will be measured by a trained research assistant.

DEGS1 aims to analyze health status, health risks and resources, functional capacity levels, and disability in the German adult population. Prevalence estimates will be obtained and time trends will be analyzed in comparison with data from the GNHIES98. Changes in health status and risk factors over time at the individual level will be studied among DEGS1 participants who already took part in the GNHIES98. Major study objectives include:
(1) To estimate the prevalence of diseases and risk factors with high public health impact and to identify differences according to socio-demographic characteristics, region of residence as well as changes over time;
(2) To analyze data on medication use and health care services utilization with regard to aspects of treatment effectiveness and quality of care;
(3) To investigate the association between mental health, physical health, and health-related behavior;
(4) To study patterns and determinants of co- and multimorbidity in the population 65 years and older in association with functional capacity levels, disability and health-related quality of life;
(5) To analyze allergic sensitization patterns and associations with manifest allergic disease in the population;
(6) To assess nutritional health risks and states of nutritional deficiency based on semiquantitative assessment of dietary habits and serum concentrations of various micronutrients (25-hydroxyvitamin D, vitamin B 12, iron, and ferritin in serum; serum and erythrocyte folate; urinary sodium excretion);
(7) To estimate iodine intake at the population level based on urinary iodine excretion;
(8) To examine individual changes in health status and cardiometabolic risk factors (e. g. body mass index; glycosylated hemoglobin A1c, serum lipids) over time.

We searched for published GWASs evaluating individuals of European ancestry on the GWAS Catalog and PubMed (the last search was performed in May 2022). We did not find any GWAS conducted for vitamins B1, B2, B3, B5, B7, sulfur, iodine, chloride, and fluoride. The GWASs conducted for vitamin K, potassium, sodium, cobalt, chromium, and molybdenum were excluded because of no significant genome-wide results [8 (link), 18 (link), 19 (link)]. In total, fourteen micronutrients of potential interest were identified: calcium [5 (link)], copper [6 (link)], iron [7 (link)], magnesium [8 (link)], selenium [6 (link)], zinc [6 (link)], beta-carotene [9 (link)], folate [10 (link)], vitamin A [11 (link)], vitamin B6 [12 (link)], vitamin B12 [10 (link)], vitamin C [13 (link)], vitamin D [14 (link)], and vitamin E [15 (link)] (Additional file 2: Additional Text) [5 (link)–15 (link), 20 , 21 (link)]. For copper, we also identified a more recent and larger GWAS by Jäger et al. [20 ], but given that this study reported Z-scores and not beta-coefficients, we used the study by Evans et al. [6 (link)] in order to improve interpretability. However, the genetic instruments from the GWAS by Jäger et al. [20 ] were used in secondary analyses. Vitamin A and vitamin E were excluded because those GWASs were adjusted for body mass index (BMI) [22 (link)] which might introduce collider bias if the genetic instruments of the exposure of interest also have an effect on BMI [23 (link)].
For the main MR analysis, we included independent SNPs (r² < 0.001 within 10,000-kb windows), strongly associated (P ≤ 5E−08) with the blood level of each micronutrient.

For an instrumental variable to be valid, three key assumptions must be met: the instrument must be robustly associated with the exposure, it cannot affect a confounder of the exposure-outcome association, and it must only affect the outcome through the risk factor [4 (link)]. Horizontal pleiotropy—that the SNP has multiple effects—can violate those assumptions. MR-Egger, weighted median, simple mode, and weighted mode are some of the most common sensitivity analyses to account for horizontal pleiotropy [17 ]. These analyses were only conducted when the number of genetic instruments was ≥ 3. The MR-Egger method allows some SNPs to affect the outcome through a pathway other than through the exposure. If the intercept term differs from zero, this indicates that not all the included instruments are valid, and the standard estimates (i.e., IVW) may be biased [39 (link)]. The weighted median method provides a valid MR estimate when up to 50% of the included instruments are invalid. This method calculates the weighted median estimate by ordering the genetic variants according to the magnitude of their estimates [40 (link)]. The mode-based methods (simple mode and weighted mode) assume that the most common causal effect is consistent with the true causal effect, allowing some instruments to be invalid without biasing the estimated causal effect [41 (link)].
To evaluate if the differences in the individual effect sizes among the genetic instruments may be related to pleiotropic effects rather than chance, we conducted Cochran’s Q statistical test [42 (link)]. This test was only conducted when two or more variants were available, and a P < 0.05 was considered significant in the test for heterogeneity.
We further evaluated whether the genetic instruments were associated with other phenotypes using PhenoScanner V2 [43 (link)], available at http://www.phenoscanner.medschl.cam.ac.uk/ (accessed 30 October 2022). Additionally, we performed leave-one-out analyses for micronutrients containing > 2 SNPs. This was performed to examine the robustness of the IVW estimates and if any specific SNP drove the association (which could be due to pleiotropy) [4 (link)].
Multivariable MR was used to evaluate whether any of the identified phenotypes on PhenoScanner had introduced bias due to pleiotropy [17 ]. The genetic variants for the potentially pleiotropic phenotypes were collected from IEU OpenGWAS [44 ]. Based on the identified potentially pleiotropic pathways, we conducted one multivariable MR analysis of copper on the risk of gastrointestinal infections where we included erythrocyte count (GWAS identifier: ukb-d-30250_irnt) and hemoglobin concentration (GWAS identifier: ebi-a-GCST004615) in the analysis.

The present study took place from September 2019 to March 2020. During the first two weeks, 127 preschool children’s parents were recruited to participate in the study according to the following inclusion and exclusion criteria.
Out of 127, 9% (n = 12) of the children was not included in the study as they either refused or did not show up to the explanatory meetings organized at T0. Thus, 115 children were enrolled from 5 preschool classes.
Parents were asked to sign the informed consent and to fill in a Food Frequency Questionnaire (FFQ) to evaluate their children’s dietary habits and physical activity. Afterwards parents, grandparents or other caregivers and children were invited together with teachers and school kitchen staff to attend an explanatory meeting to learn how to use “Nutripiatto” and its guide (T0). The explanatory meetings were held by a nutritionist and organized several times for small groups of people (8 persons), accordingly to the time in which parents, grandparents or other caregivers, teachers and school staff were recruited. To allow parents, grandparents or other caregivers to participate, completion of the questionnaire and the explanatory meetings took place at school entrance or exit times.
During the meeting, the importance of the right amount of nutrients and micronutrients in the diet as well as the proper portion sizes was emphasized. Parents, grandparents or other caregivers were recommended to read the explanatory guide and to try cooking the recipes with their children to improve their food choices and healthy eating habits. They also had the opportunity to meet the nutritionist after the explanatory meeting if they need further information on “Nutripiatto” or issues related to childhood nutrition. About 10% of the parents wanted a further meeting with the nutritionist to better understand the project and what was expected of them. The food education interventions for children were composed by two interactive sessions on healthy dietary habits and lifestyle tailored to the children’s age. They were mainly interactive interventions with games and practical activities to encourage active learning. Children played with food-shaped toys to learn about the main nutrients they contained. They built a Mediterranean diet food pyramid with printable word scrambles, using flash cards or food group colouring sheets. Nutritionists explained and showed children how to use “Nutripiatto” correctly. At the end of each session, children were asked to draw foods and put them in their “Nutripiatto” to evaluate how much they had learned on food composition and healthy eating.
They were invited to bring their “Nutripiatto” at home, while parents were asked to allow their children to use it for their main meals for a month.
At the end of the one-month period (T1), parents were asked to fill in a second FFQ to evaluate changes in their children’s eating habits compared to baseline.
The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee Campus Bio-Medico University of Rome for studies involving humans (approval code of the study 131/21).