Erik A. Ehli

The Young Netherlands Twin Register (YNTR): Longitudinal Twin and Family Studies in Over 70,000 Children

The Netherlands Twin Register (NTR) began in 1987 with data collection in twins and their families, including families with newborn twins and triplets. Twenty-five years later, the NTR has collected at least one survey for 70,784 children, born after 1985. For the majority of twins, longitudinal data collection has been done by age-specific surveys. Shortly after giving birth, mothers receive a first survey with items on pregnancy and birth. At age 2, a survey on growth and achievement of milestones is sent. At ages 3, 7, 9/10, and 12 parents and teachers receive a series of surveys that are targeted at the development of emotional and behavior problems. From age 14 years onward, adolescent twins and their siblings report on their behavior problems, health, and lifestyle. When the twins are 18 years and older, parents are also invited to take part in survey studies. In sub-groups of different ages, in-depth phenotyping was done for IQ, electroencephalography , MRI, growth, hormones, neuropsychological assessments, and cardiovascular measures. DNA and biological samples have also been collected and large numbers of twin pairs and parents have been genotyped for zygosity by either micro-satellites or sets of short nucleotide polymorphisms and repeat polymorphisms in candidate genes. Subject recruitment and data collection is still ongoing and the longitudinal database is growing. Data collection by record linkage in the Netherlands is beginning and we expect these combined longitudinal data to provide increased insights into the genetic etiology of development of mental and physical health in children and adolescents.

Genetic and environmental influences interact with age and sex in shaping the human methylome

The methylome is subject to genetic and environmental effects. Their impact may depend on sex and age, resulting in sex- and age-related physiological variation and disease susceptibility. Here we estimate the total heritability of DNA methylation levels in whole blood and estimate the variance explained by common single nucleotide polymorphisms at 411,169 sites in 2,603 individuals from twin families, to establish a catalogue of between-individual variation in DNA methylation. Heritability estimates vary across the genome (mean=19%) and interaction analyses reveal thousands of sites with sex-specific heritability as well as sites where the environmental variance increases with age. Integration with previously published data illustrates the impact of genome and environment across the lifespan at methylation sites associated with metabolic traits, smoking and ageing. These findings demonstrate that our catalogue holds valuable information on locations in the genome where methylation variation between people may reflect disease-relevant environmental exposures or genetic variation.

The antisense RNA of the par locus of pAD1 regulates the expression of a 33‐amino‐acid toxic peptide by an unusual mechanism

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA‐regulated post‐segregational killing system (PSK) identified in a Gram‐positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA I encodes an open reading frame of 33 codons designated fst. The results presented here demonstrate that the peptide encoded by fst is the par toxin. The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the fst‐encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem–loop structure at the 5′ end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300‐fold increase in production of the fst‐encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of fst in plasmid‐free cells.

Minimally invasive genomic and transcriptomic profiling of visceral cancers by next-generation sequencing of circulating exosomes

High-resolution profiling of the genomic and transcriptomic landscapes of visceral cancers using the DNA and RNA of tumor shed exosomes has the potential to be used as a clinical tool for cancer diagnosis, therapeutic stratification and treatment monitoring, precluding the need for direct tumor sampling.

Lipid-lowering effect of berberine in human subjects and rats.

Due to serious adverse effects and the limited effectiveness of currently available pharmacological therapies for obesity, many research efforts have focused on the development of drugs from natural products. Our previous studies demonstrated that berberine, an alkaloid originally isolated from traditional Chinese herbs, prevented fat accumulation in vitro and in vivo. In this pilot study, obese human subjects (Caucasian) were given 500 mg berberine orally three times a day for twelve weeks. The efficacy and safety of berberine treatment was determined by measurements of body weight, comprehensive metabolic panel, blood lipid and hormone levels, expression levels of inflammatory factors, complete blood count, and electrocardiograph. A Sprague-Dawley rat experiment was also performed to identify the anti-obesity effects of berberine treatment. The results demonstrate that berberine treatment produced a mild weight loss (average 5 lb/subject) in obese human subjects. But more interestingly, the treatment significantly reduced blood lipid levels (23% decrease of triglyceride and 12.2% decrease of cholesterol levels) in human subjects. The lipid-lowering effect of berberine treatment has also been replicated in the rat experiment (34.7% decrease of triglyceride and 9% decrease of cholesterol level). Cortisol, calcitriol, ACTH, TSH, FT4, and SHBG levels were not significantly changed following 12 weeks of berberine treatment. However, there was interestingly, an increase in calcitriol levels seen in all human subjects following berberine treatment (mean 59.5% increase, p=0.11). Blood inflammatory factors (CRP, IL-6, TNFα, COX-2) and erythrocyte sedimentation rate (ESR) were not significantly affected by treatment with berberine. Tests of hematological, cardiovascular, liver, and kidney function following berberine treatment showed no detrimental side effects to this natural compound. Collectively, this study demonstrates that berberine is a potent lipid-lowering compound with a moderate weight loss effect, and may have a possible potential role in osteoporosis treatment/prevention.

De novo and inherited CNVs in MZ twin pairs selected for discordance and concordance on Attention Problems

Copy number variations (CNVs) have been reported to be causal suspects in a variety of psychopathologic traits. We investigate whether de novo and/or inherited CNVs contribute to the risk for Attention Problems (APs) in children. Based on longitudinal phenotyping, 50 concordant and discordant monozygotic (MZ) twin pairs were selected from a sample of ∼3200 MZ pairs. Two types of de novo CNVs were investigated: (1) CNVs shared by both MZ twins, but not inherited (pre-twinning de novo CNVs), which were detected by comparing copy number (CN) calls between parents and twins and (2) CNVs not shared by co-twins (post-twinning de novo CNVs), which were investigated by comparing the CN calls within MZ pairs. The association between the overall CNV burden and AP was also investigated for CNVs genome-wide, CNVs within genes and CNVs outside of genes. Two de novo CNVs were identified and validated using quantitative PCR: a pre-twinning de novo duplication in a concordant-unaffected twin pair and a post-twinning deletion in the higher scoring twin from a concordant-affected pair. For the overall CNV burden analyses, affected individuals had significantly larger CNVs that overlapped with genes than unaffected individuals (P=0.008). This study suggests that the presence of larger CNVs may increase the risk for AP, because they are more likely to affect genes, and confirms that MZ twins are not always genetically identical.

COMT Val158Met Genotype as a Risk Factor for Problem Behaviors in Youth

OBJECTIVE To test the association between the catechol-O-methyltransferase (COMT) Val158Met polymorphism and both aggressive behavior and attention problems in youth. We hypothesized that youth carrying a Met allele would have greater average aggressive behavior scores, and that youth exhibiting Val-homozygosity would have greater average attention problems scores. METHOD Complete data on maternally rated Child Behavior Checklist (CBCL) measures of aggressive behavior (AGG) and attention problems (AP), COMT polymorphism data, demographics, and maternal parenting quality were available for 149 youth (6 to 18 years old). Multivariable linear regression models were used to test the degree to which youth COMT Val158Met genotype was associated with AGG and AP while statistically controlling for age, gender, parental socioeconomic status (SES), and maternal parenting quality from the Alabama Parenting Questionnaire. RESULTS Mothers of Met-carriers rated their children higher on average AGG scores when compared with mothers of Val-homozygotes (p = .016). Further analyses revealed that this association was even more robust for maternal ratings of direct aggression (p = .007). The hypothesized association between Val-homozygosity and higher average AP scores relative to average AP scores of Met-carriers did not quite reach statistical significance (p = .062). CONCLUSIONS After controlling for demographics, SES, and maternal parenting quality as confounders, there remains a strong association between youth carrying a Met allele and higher average AGG scores relative to Val-homozygotes.

Epigenetic Variation in Monozygotic Twins: A Genome-Wide Analysis of DNA Methylation in Buccal Cells

DNA methylation is one of the most extensively studied epigenetic marks in humans. Yet, it is largely unknown what causes variation in DNA methylation between individuals. The comparison of DNA methylation profiles of monozygotic (MZ) twins offers a unique experimental design to examine the extent to which such variation is related to individual-specific environmental influences and stochastic events or to familial factors (DNA sequence and shared environment). We measured genome-wide DNA methylation in buccal samples from ten MZ pairs (age 8–19) using the Illumina 450k array and examined twin correlations for methylation level at 420,921 CpGs after QC. After selecting CpGs showing the most variation in the methylation level between subjects, the mean genome-wide correlation (rho) was 0.54. The correlation was higher, on average, for CpGs within CpG islands (CGIs), compared to CGI shores, shelves and non-CGI regions, particularly at hypomethylated CpGs. This finding suggests that individual-specific environmental and stochastic influences account for more variation in DNA methylation in CpG-poor regions. Our findings also indicate that it is worthwhile to examine heritable and shared environmental influences on buccal DNA methylation in larger studies that also include dizygotic twins.

Enterococcus faecalis Plasmid pAD1-Encoded Fst Toxin Affects Membrane Permeability and Alters Cellular Responses to Lantibiotics

Fst is a peptide toxin encoded by the par toxin-antitoxin stability determinant of Enterococcus faecalis plasmid pAD1. Intracellular overproduction of Fst resulted in simultaneous inhibition of all cellular macromolecular synthesis concomitant with cell growth inhibition and compromised the integrity of the cell membrane. Cells did not lyse or noticeably leak intracellular contents but had specific defects in chromosome partitioning and cell division. Extracellular addition of synthetic Fst had no effect on cell growth. Spontaneous Fst-resistant mutants had a phenotype consistent with changes in membrane composition. Interestingly, overproduction of Fst sensitized cells to the lantibiotic nisin, and Fst-resistant mutants were cross-resistant to nisin and the pAD1-encoded cytolysin.

Genetic Basis of a Cognitive Complexity Metric

Relational complexity (RC) is a metric reflecting capacity limitation in relational processing. It plays a crucial role in higher cognitive processes and is an endophenotype for several disorders. However, the genetic underpinnings of complex relational processing have not been investigated. Using the classical twin model, we estimated the heritability of RC and genetic overlap with intelligence (IQ), reasoning, and working memory in a twin and sibling sample aged 15-29 years (N = 787). Further, in an exploratory search for genetic loci contributing to RC, we examined associated genetic markers and genes in our Discovery sample and selected loci for replication in four independent samples (ALSPAC, LBC1936, NTR, NCNG), followed by meta-analysis (N>6500) at the single marker level. Twin modelling showed RC is highly heritable (67%), has considerable genetic overlap with IQ (59%), and is a major component of genetic covariation between reasoning and working memory (72%). At the molecular level, we found preliminary support for four single-marker loci (one in the gene DGKB), and at a gene-based level for the NPS gene, having influence on cognition. These results indicate that genetic sources influencing relational processing are a key component of the genetic architecture of broader cognitive abilities. Further, they suggest a genetic cascade, whereby genetic factors influencing capacity limitation in relational processing have a flow-on effect to more complex cognitive traits, including reasoning and working memory, and ultimately, IQ.