Claudia J. Kruithof

The Generation R Study: Biobank update 2015

The Generation R Study is a population-based prospective cohort study from fetal life until adulthood. The study is designed to identify early environmental and genetic causes and causal pathways leading to normal and abnormal growth, development and health from fetal life, childhood and young adulthood. In total, 9,778 mothers were enrolled in the study. Data collection in children and their parents include questionnaires, interviews, detailed physical and ultrasound examinations, behavioural observations, Magnetic Resonance Imaging and biological samples. Efforts have been conducted for collecting biological samples including blood, hair, faeces, nasal swabs, saliva and urine samples and generating genomics data on DNA, RNA and microbiome. In this paper, we give an update of the collection, processing and storage of these biological samples and available measures. Together with detailed phenotype measurements, these biological samples provide a unique resource for epidemiological studies focused on environmental exposures, genetic and genomic determinants and their interactions in relation to growth, health and development from fetal life onwards.

Meta-analysis of genome-wide scans for total body BMD in children and adults reveals allelic heterogeneity and age-specific effects at the WNT16 locus

To identify genetic loci influencing bone accrual, we performed a genome-wide association scan for total-body bone mineral density (TB-BMD) variation in 2,660 children of different ethnicities. We discovered variants in 7q31.31 associated with BMD measurements, with the lowest P = 4.1×10−11 observed for rs917727 with minor allele frequency of 0.37. We sought replication for all SNPs located ±500 kb from rs917727 in 11,052 additional individuals from five independent studies including children and adults, together with de novo genotyping of rs3801387 (in perfect linkage disequilibrium (LD) with rs917727) in 1,014 mothers of children from the discovery cohort. The top signal mapping in the surroundings of WNT16 was replicated across studies with a meta-analysis P = 2.6×10−31 and an effect size explaining between 0.6%–1.8% of TB-BMD variance. Conditional analyses on this signal revealed a secondary signal for total body BMD (P = 1.42×10−10) for rs4609139 and mapping to C7orf58. We also examined the genomic region for association with skull BMD to test if the associations were independent of skeletal loading. We identified two signals influencing skull BMD variation, including rs917727 (P = 1.9×10−16) and rs7801723 (P = 8.9×10−28), also mapping to C7orf58 (r2 = 0.50 with rs4609139). Wnt16 knockout (KO) mice with reduced total body BMD and gene expression profiles in human bone biopsies support a role of C7orf58 and WNT16 on the BMD phenotypes observed at the human population level. In summary, we detected two independent signals influencing total body and skull BMD variation in children and adults, thus demonstrating the presence of allelic heterogeneity at the WNT16 locus. One of the skull BMD signals mapping to C7orf58 is mostly driven by children, suggesting temporal determination on peak bone mass acquisition. Our life-course approach postulates that these genetic effects influencing peak bone mass accrual may impact the risk of osteoporosis later in life.

Parental smoking during pregnancy, early growth, and risk of obesity in preschool children: the Generation R Study

BACKGROUND Maternal smoking during pregnancy seems to be associated with obesity in offspring. Not much is known about the specific critical exposure periods or underlying mechanisms for this association. OBJECTIVE We assessed the associations of active maternal and paternal smoking during pregnancy with early growth characteristics and risks of overweight and obesity in preschool children. DESIGN This study was a population-based, prospective cohort study from early fetal life until the age of 4 y in 5342 mothers and fathers and their children. Growth characteristics [head circumference, length, weight, and body mass index (BMI; in kg/m(2))] and overweight and obesity were repeatedly measured at the ages of 1, 2, 3, and 4 y. RESULTS In comparison with children from nonsmoking mothers, children from mothers who continued smoking during pregnancy had persistently smaller head circumferences and heights until the age of 4 y, whereas their weights were lower only until the age of 3 mo. This smaller length and normal to higher weight led to an increased BMI [SD score difference: 0.11; 95% CI: 0.02, 0.20; P < 0.05)] and an increased risk of obesity (odds ratio: 1.61; 95% CI: 1.03, 2.53; P < 0.05) at the age of 4 y. In nonsmoking mothers, paternal smoking was not associated with postnatal growth characteristics or risk of obesity in offspring. Maternal smoking during pregnancy was associated with a higher BMI at the age of 4 y in children with a normal birth weight and in those who were small for gestational age at birth. CONCLUSION Our findings suggest that direct intrauterine exposure to smoke until late pregnancy leads to different height and weight growth adaptations and increased risks of overweight and obesity in preschool children.

Maternal anthropometrics are associated with fetal size in different periods of pregnancy and at birth. The Generation R Study.

Objective  We aimed to examine the associations of maternal anthropometrics with fetal weight measured in different periods of pregnancy and with birth outcomes.

Phenotypic Dissection of Bone Mineral Density Reveals Skeletal Site Specificity and Facilitates the Identification of Novel Loci in the Genetic Regulation of Bone Mass Attainment

Heritability of bone mineral density (BMD) varies across skeletal sites, reflecting different relative contributions of genetic and environmental influences. To quantify the degree to which common genetic variants tag and environmental factors influence BMD, at different sites, we estimated the genetic (rg) and residual (re) correlations between BMD measured at the upper limbs (UL-BMD), lower limbs (LL-BMD) and skull (SK-BMD), using total-body DXA scans of ∼4,890 participants recruited by the Avon Longitudinal Study of Parents and their Children (ALSPAC). Point estimates of rg indicated that appendicular sites have a greater proportion of shared genetic architecture (LL-/UL-BMD rg = 0.78) between them, than with the skull (UL-/SK-BMD rg = 0.58 and LL-/SK-BMD rg = 0.43). Likewise, the residual correlation between BMD at appendicular sites (re = 0.55) was higher than the residual correlation between SK-BMD and BMD at appendicular sites (re = 0.20–0.24). To explore the basis for the observed differences in rg and re, genome-wide association meta-analyses were performed (n∼9,395), combining data from ALSPAC and the Generation R Study identifying 15 independent signals from 13 loci associated at genome-wide significant level across different skeletal regions. Results suggested that previously identified BMD-associated variants may exert site-specific effects (i.e. differ in the strength of their association and magnitude of effect across different skeletal sites). In particular, variants at CPED1 exerted a larger influence on SK-BMD and UL-BMD when compared to LL-BMD (P = 2.01×10−37), whilst variants at WNT16 influenced UL-BMD to a greater degree when compared to SK- and LL-BMD (P = 2.31×10−14). In addition, we report a novel association between RIN3 (previously associated with Pagets disease) and LL-BMD (rs754388: β = 0.13, SE = 0.02, P = 1.4×10−10). Our results suggest that BMD at different skeletal sites is under a mixture of shared and specific genetic and environmental influences. Allowing for these differences by performing genome-wide association at different skeletal sites may help uncover new genetic influences on BMD.

Challenges in conducting genome-wide association studies in highly admixed multi-ethnic populations: the Generation R Study

Genome-wide association studies (GWAS) have been successful in identifying loci associated with a wide range of complex human traits and diseases. Up to now, the majority of GWAS have focused on European populations. However, the inclusion of other ethnic groups as well as admixed populations in GWAS studies is rapidly rising following the pressing need to extrapolate findings to non-European populations and to increase statistical power. In this paper, we describe the methodological steps surrounding genetic data generation, quality control, study design and analytical procedures needed to run GWAS in the multiethnic and highly admixed Generation R Study, a large prospective birth cohort in Rotterdam, the Netherlands. Furthermore, we highlight a number of practical considerations and alternatives pertinent to the quality control and analysis of admixed GWAS data.

Prevalence and predictors of vitamin D deficiency based on maternal mid-gestation and neonatal cord bloods: The Generation R Study

BACKGROUND Population-based studies have confirmed that the prevalence of vitamin D deficiency is substantial in many societies, and is of particular concern in pregnant women. Vitamin D deficiency during pregnancy is associated with a wide range of adverse maternal and offspring health outcomes. To date, studies of vitamin D deficiency during pregnancy have focused on measurements at one or two time points in isolation. We examined both midgestation and cord blood 25 hydroxyvitamin D (25OHD) concentration and explored the prevalence and correlates of vitamin D deficiency in a large ethnically diverse cohort of pregnant women and their infants in the Netherlands. METHODS This study was embedded in the Generation R Study, a population-based prospective cohort from fetal life onwards in Rotterdam, The Netherlands. Using a highly sensitive tandem mass spectroscopy-based assay, we measured 25OHD in 7256 midgestation samples (mean gestation 20.6 weeks) and 5023 neonatal cord blood samples (mean gestation 40.0 weeks). Using a conservative threshold of less than 25nmol/L to define vitamin D deficiency, we examined the prevalence and socio-demographic correlates of vitamin D deficiency in mothers and infants. We also derived a measure of vitamin D deficiency based on the two time points in order to explore persistent vitamin D deficiency in mother-infant pairs. RESULTS The prevalence of vitamin D deficiency at midgestation was 26%, while in neonates 46% were deficient. 21% of the mother-infant pairs had persistent vitamin D deficiency (i.e., deficient in maternal and cord samples) and an additional 29% were vitamin D deficient in one of the two samples only. Persistent vitamin D deficiency was strongly associated with non-European ancestry and spring birth. CONCLUSIONS A sizeable proportion of women and their neonatal offspring in the Generation R cohort were vitamin D deficient. In light of the large body of evidence linking vitamin D deficiency with adverse health outcomes for pregnant women and their offspring, our findings indicate a large unmet need in this population. In particular, women and infants from non-European ethnic background are at high risk of vitamin D deficiency.

Assessment of maternal smoking status during pregnancy and the associations with neonatal outcomes.

INTRODUCTION Single assessment of smoking during pregnancy may lead to misclassification due to underreporting or failure of smoking cessation. We examined the percentage of mothers who were misclassified in smoking status based on single assessment, as compared with repeated assessment, and whether this misclassification leads to altered effect estimates for the associations between maternal smoking and neonatal complications. METHODS This study was performed in 5,389 mothers participating in a prospective population-based cohort study in the Netherlands. Smoking status was assessed 3 times during pregnancy using questionnaires. Information on birth weight and neonatal complications was obtained from hospital records. RESULTS For categorizing mothers per smoking status, Cohens Kappa coefficient was .86 (p < .001) between single and repeated assessments. Of all mothers who reported nonsmoking or first trimester-only smoking in early pregnancy, 1.7% (70 of 4,141) and 33.7% (217 of 643), respectively, were reclassified to continued smoking based on repeated assessment. Younger, shorter lower educated mothers who had non-European ethnicity experienced more stress, consumed more alcohol, and did not use folic acid supplements had higher risk of underreporting their smoking status or failure of smoking cessation. Marginal differences were found on the associations of maternal smoking with neonatal complications between single or repeated assessment. CONCLUSIONS Our results suggest that single assessment of smoking during pregnancy leads to underestimation of the continued smoking prevalence, especially among mothers who reported quitting smoking in first trimester. However, this underestimation does not materially change the effect estimates for the associations between maternal smoking and neonatal outcomes.

Gestational vitamin D deficiency and autism-related traits: the Generation R Study

There is intense interest in identifying modifiable risk factors associated with autism-spectrum disorders (ASD). Autism-related traits, which can be assessed in a continuous fashion, share risk factors with ASD, and thus can serve as informative phenotypes in population-based cohort studies. Based on the growing body of research linking gestational vitamin D deficiency with altered brain development, this common exposure is a candidate modifiable risk factor for ASD and autism-related traits. The association between gestational vitamin D deficiency and a continuous measure of autism-related traits at ~6 years (Social Responsiveness Scale; SRS) was determined in a large population-based cohort of mothers and their children (n=4229). 25-hydroxyvitamin D (25OHD) was assessed from maternal mid-gestation sera and from neonatal sera (collected from cord blood). Vitamin D deficiency was defined as 25OHD concentrations less than 25 nmol l−1. Compared with the 25OHD sufficient group (25OHD>50 nmol l−1), those who were 25OHD deficient had significantly higher (more abnormal) SRS scores (mid-gestation n=2866, β=0.06, P<0.001; cord blood n=1712, β=0.03, P=0.01). The findings persisted (a) when we restricted the models to offspring with European ancestry, (b) when we adjusted for sample structure using genetic data, (c) when 25OHD was entered as a continuous measure in the models and (d) when we corrected for the effect of season of blood sampling. Gestational vitamin D deficiency was associated with autism-related traits in a large population-based sample. Because gestational vitamin D deficiency is readily preventable with safe, cheap and accessible supplements, this candidate risk factor warrants closer scrutiny.

Early childhood growth patterns and school-age respiratory resistance, fractional exhaled nitric oxide and asthma

Greater infant weight gain is associated with lower lung function and increased risk of childhood asthma. The role of early childhood peak growth patterns is unclear. We assessed the associations of individually derived early childhood peak growth patterns with respiratory resistance, fractional exhaled nitric oxide, wheezing patterns, and asthma until school‐age.