N Y Souren

Assisted reproductive technologies do not enhance the variability of DNA methylation imprints in human

Background Assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) are believed to destabilise genomic imprints. An increased frequency of Beckwith–Wiedemann syndrome in children born after ART has been reported. Other, mostly epidemiological, studies argue against this finding. Objective To examine the effect of ART on the stability of DNA methylation imprints, DNA was extracted from maternal peripheral blood (MPB), umbilical cord blood (UCB) and amnion/chorion tissue (ACT) of 185 phenotypically normal children (77 ICSI, 35 IVF, and 73 spontaneous conceptions). Using bisulfite based technologies 10 differentially methylated regions (DMRs) were analysed, including KvDMR1, H19, SNRPN, MEST, GRB10, DLK1/MEG3 IG-DMR, GNAS NESP55, GNAS NESPas, GNAS XL-alpha-s and GNAS Exon1A. Results Methylation indices (MI) do not reveal any significant differences at nine DMRs among the conception groups in neither MPB, UCB nor in ACT. The only slightly variable DMR was that of MEST. Here the mean MI was higher in UCB and MPB of IVF cases (mean MI±SD: 0.41±0.03 (UCB) and 0.40±0.03 (MPB)) compared to the ICSI (0.38±0.03, p=0.003 (UCB); 0.37±0.04, p=0.0007 (MPB)) or spontaneous cases (0.38±0.03, p=0.003 (UCB); 0.38±0.04, p=0.02 (MPB)). Weak but suggestive correlations between DMRs were, however, found between MPB, UCB and ACT. Conclusion This study supports the notion that children conceived by ART do not show a higher degree of imprint variability and hence do not have an a priori higher risk for imprinting disorders.

Carbamoyl Phosphate Synthetase Polymorphisms as a Risk Factor for Necrotizing Enterocolitis

A C-to-A nucleotide transversion (T1405N) in the gene that encodes carbamoyl-phosphate synthetase 1 (CPS1) has been correlated with low plasma concentrations of l-arginine in neonates. As plasma l-arginine concentrations are decreased in premature infants with necrotizing enterocolitis (NEC), we hypothesized that the CPS1 T1405N polymorphism would correlate with the presence of NEC. We analyzed the CPS1 genotypes for the T1405N polymorphism in 17 preterm infants (≤30 wk and <1500 g) with established NEC, 34 preterm infants without NEC, and 25 healthy term infants. Distribution of genotypes did not differ between the NEC population (CC:AC:AA = 70.6%:23.5%:5.9%) and the preterm control group (CC:AC:AA = 41.2%:35.3%:23.5%; p = 0.110) or the term group (CC:AC:AA = 44%:48%:8%; p = 0.228). The C allele frequency was 82.4% in NEC and 58.8% in preterm control infants (p = 0.018) and analysis for linear trend demonstrated that incidence of NEC increased with the number of C alleles (p = 0.037). The CC genotype was associated with an increased risk of NEC in the preterm infants [odds ratio (OR) = 3.43, 95% confidence interval (CI): 1.01–11.49, p = 0.048), when compared with the grouped together AA/AC genotypes. These data suggest that the CPS1 T1405N polymorphism may be associated with the risk of NEC in preterm infants.

Common SNPs in LEP and LEPR associated with birth weight and type 2 diabetes-related metabolic risk factors in twins

Objective:Children born small for gestational age are at increased risk of developing type 2 diabetes in adulthood. The satiety signal leptin that regulates food intake and energy expenditure might be a possible molecular link, as umbilical cord leptin levels are positively correlated with birth weight. In the present study, we examined whether common single nucleotide polymorphisms (SNPs) in the leptin (LEP; 19G>A) gene and its receptor (LEPR; Q223R and K109R) are associated with birth weight and adult metabolic risk factors for type 2 diabetes in twins.Design:SNPs were genotyped in 396 monozygotic and 232 dizygotic twins (286 men and 342 women, mean age 25 years) recruited from the East Flanders Prospective Twin Survey. Data were analysed using linear mixed models.Results:The LEPR K109R SNP was associated with birth weight (KK, KR and RR (95% confidence interval, CI): 2511 (2465–2557), 2575 (2516–2635) and 2726 (2606–2845) gram; Padditive=0.001). Also the LEPR Q223R SNP showed a significant association with weight at birth (QQ, QR and RR (95% CI): 2492 (2431–2554), 2545 (2495–2595) and 2655 (2571–2740) gram; Padditive=0.003). Furthermore, an interaction between the LEPR K109R and the Q223R SNP on birth weight was observed (P=0.014). G allele carriers of the LEP 19G>A SNP had higher high-density lipoprotein (HDL) cholesterol levels compared to 19A homozygotes (GX vs AA (95% CI): 1.62 (1.58–1.66) vs 1.49 (1.40–1.58) mmol l−1; Precessive=0.013).Conclusions:This study indicates that leptin may act as a growth-promoting signal during fetal development, and suggests a possible role for the LEPR in explaining the inverse relationship between birth weight and the development of metabolic diseases in adulthood. Additionally, these results suggest that the LEP 19G>A SNP affect HDL cholesterol levels.

Adult monozygotic twins discordant for intra-uterine growth have indistinguishable genome-wide DNA methylation profiles

BackgroundLow birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intra-uterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. To address this, we acquire genome-wide DNA methylation profiles from saliva DNA in a unique cohort of 17 monozygotic monochorionic female twins very discordant for birth weight. We examine if adverse prenatal growth conditions experienced by the smaller co-twins lead to long-lasting DNA methylation changes.ResultsOverall, co-twins show very similar genome-wide DNA methylation profiles. Since observed differences are almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3,153 are differentially methylated between the heavy and light co-twins at nominal significance, of which 45 show sensible absolute mean β-value differences. Deep bisulfite sequencing of eight such loci reveals that differences remain in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicates no significant intra-pair differences.ConclusionsSevere intra-uterine growth differences observed within these monozygotic twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies.

DNA Methylation Variability at Growth-Related Imprints Does not Contribute to Overweight in Monozygotic Twins Discordant for BMI

Defective genomic imprinting is often associated with syndromes that include abnormal growth as a clinical phenotype. However, whether differential methylation at imprinted loci also contributes to nonsyndromic abnormal body weight regulation is yet unknown. In this study, we investigated a potential contribution of aberrant DNA methylation at nine differentially methylated regions (DMRs) to the development of nonsyndromic overweight. Sixteen monozygotic (MZ) twins discordant for BMI (BMI difference ranging from 2.9–9.5 kg/m2) were recruited from the East Flanders Prospective Twin Survey. DNA extracted from saliva samples was bisulfite‐treated followed by PCR amplification of target regions in DMRs most representative for abnormal growth syndromes: KvDMR1, H19 CTCF4, H19 CTCF6, IGF2 DMR0, IGF2 DMR2, GRB10, MEST, SNRPN, GNAS XL‐α‐s and GNAS Exon1A. At the DMRs analyzed, methylation‐dependent primer extension experiments revealed only small intrapair differences in methylation indexes (MI) between the heavy and lean co‐twins. In addition, no significant correlations between intrapair BMI differences and intrapair differences in MI were observed. In conclusion, DNA methylation variability at the nine DMRs analyzed does not seem to contribute to the discordancy in BMI observed in these MZ twins.

DNA methylation studies on imprinted loci in a male monozygotic twin pair discordant for Beckwith–Wiedemann syndrome

Tierling S, Souren NY, Reither S, Zang KD, Meng‐Hentschel J, Leitner D, Oehl‐Jaschkowitz B, Walter J. DNA methylation studies on imprinted loci in a male monozygotic twin pair discordant for Beckwith–Wiedemann syndrome.

Genetics of maximally attained lung function: A role for leptin?

OBJECTIVES To estimate the heritabilities of maximally attained lung function in young adult twins, and to examine whether circulating leptin, leptin (LEP) and leptin receptor (LEPR) gene polymorphisms are associated with maximally attained lung function. METHODS Measures on forced expiratory volume in 1 s (FEV(1)) and forced vital capacity (FVC) were available of 578 twins recruited from the East Flanders Prospective Twin Survey (165 monozygotic (MZ) and 73 dizygotic (DZ) complete pairs and 102 single twins). Twin model fitting and (genetic) association analyses were performed. RESULTS Intra-pair correlations of FEV(1) and FVC did not differ significantly between MZ monochorionic and MZ dichorionic pairs. Heritability estimates of FEV(1) and FVC were 69% and 63%, respectively. The A allele of the LEP 19G>A SNP was significantly associated with a lower FEV(1) (p(Additive) = 0.01) and FVC (p(Dominant) = 0.047), while the LEPR K109R and Q223R SNPs showed no associations. Accounting for body mass index, serum leptin was negatively associated with FVC (p = 0.02) in men, but not in women. CONCLUSIONS More than 60% of variation in maximally attained FEV(1) and FVC is explained by genetic factors. Moreover, these results suggest that leptin may be important in the determination of maximally attainable lung function.

Parent-of-origin specific linkage and association of the IGF2 gene region with birth weight and adult metabolic risk factors.

Objective:The maternally imprinted insulin-like growth factor 2 (IGF2) gene is an important fetal growth factor and is also suggested to have postnatal metabolic effects. In this study, we examined whether common polymorphisms in IGF2 (6815_6819delAGGGC, 1156T>C and 820G>A (ApaI)) and a microsatellite marker in the close vicinity of IGF2 were linked to or associated with birth weight and adult metabolic risk factors.Design and participants:Polymorphisms were genotyped in 199 monozygotic complete twin pairs, 109 dizygotic complete twin pairs, 15 single twins, 231 mothers and 228 fathers recruited from the East Flanders Prospective Twin Survey. Conventional and parent-of-origin specific linkage and association analyses were carried out with birth weight, adult body height and parameters quantifying obesity, insulin sensitivity and dyslipidaemia measured at adult age (mean age 25 years).Results:In the parent-of-origin specific association analysis, in which only the paternally inherited allele was incorporated, the 1156T>C SNP (single nucleotide polymorphism) showed significant association with IGF-binding protein 1 (IGFBP1) levels (T and C (mean (95% CI)): 13.2 (12.1–14.3) and 16.2 (14.6–18.0) ng ml−1, P=0.002). No linkage was observed in either the conventional or in the parent-of-origin specific linkage analysis.Conclusion:This study suggests that paternally inherited alleles of a common polymorphism in the IGF2 gene affect IGFBP1 levels.

Anthropometry, carbohydrate and lipid metabolism in the East Flanders Prospective Twin Survey: linkage of candidate genes using two sib-pair based variance components analyses.

Insulin resistance and obesity are underlying causes of type 2 diabetes and therefore much interest is focused on the potential genes involved. A series of anthropometric and metabolic characteristic were measured in 240 MZ and 112 DZ twin pairs recruited from the East Flanders Prospective Twin Survey. Microsatellite markers located close to ABCC8, ADIPOQ, GCK, IGF1, IGFBP1, INSR, LEP, LEPR, PPARgamma and the RETN gene were genotyped. Univariate single point variance components linkage analyses were performed using two methods: (1) the standard method, only comprising the phenotypic and genotypic data of the DZ twin pairs and (2) the extended method, also incorporating the phenotypic data of the MZ twin pairs. Suggestive linkages (LOD > 1) were observed between the ABCC8 marker and waist-to-hip ratio and HDL-cholesterol levels. Both markers flanking ADIPOQ showed suggestive linkage with triglycerides levels, the upstream marker also with body mass and HDL-cholesterol levels. The IGFBP1 marker showed suggestive linkage with fat mass, fasting insulin and leptin levels and the LEP marker showed suggestive linkage with birth weight. This study suggests that DNA variants in ABCC8, ADIPOQ, IGFBP1 and LEP gene region may predispose to type 2 diabetes. In addition, the two methods used to perform linkage analyses yielded similar results. This was however not the case for birth weight where chorionicity seems to be an important confounder.

Association between the IL6-174G/C SNP and maximally attained lung function

Recently, He et al 1 reported that the C allele of the interleukin 6 ( IL6 )-174G/C promoter polymorphism was associated with a rapid decline in forced expiratory volume in 1 s (FEV1) (≥3% predicted/year) and susceptibility to chronic obstructive pulmonary disease (COPD) in middle-aged smokers derived from three different cohorts.1 Alternatively, as also suggested by He et al in their discussion,1 we hypothesised that the IL6 -174G/C single nucleotide polymorphism (SNP) might not only affect the rate of lung function decline but also …