Background: There are many reasons to think that epigenetics is a key determinant of fetal growth variability across the normal population. Since IGF1 and INS genes are major determinants of intrauterine growth, we examined the methylation of selected CpGs located in the regulatory region of these two genes. Methods: Cord blood was sampled in 159 newborns born to mothers prospectively followed during their pregnancy. A 142-item questionnaire was filled by mothers at inclusion, during the last trimester of the pregnancy and at the delivery. The methylation of selected CpGs located in the promoters of the IGF1 and INS genes was measured in cord blood mononuclear cells collected at birth using bisulfite-PCR-pyrosequencing. −4 Results: Methylation at IGF1 CpG-137 correlated negatively with birth length (r =0.27, P =3.5 ×10 ). The same effect size was found after adjustment for maternal age, parity, and smoking: a 10% increase in CpG-137 methylation was associated with a decrease of length by 0.23 SDS. Conclusion: The current results suggest that the methylation of IGF1 CpG-137 contributes to the individual variation of fetal growth by regulating IGF1 expression in fetal tissues. Keywords: Newborn, Growth, Epigenetics, DNA methylation Background In animal studies, IGF1 gene ablation reduces fetal Since human neonates are about the same size as the weight [2, 3]. Fetal IGF1 promotes tissue growth by birth canal, size at birth has to be a tightly regulated stimulating anabolic events and DNA synthesis. Circu- trait. It is controlled by a number of interacting genetic lating IGF1 is most often decreased in animal models of and environmental factors. Among these factors, IGF1 fetal growth restriction, as well as in human fetal growth (insulin-like growth factor-1), IGF2 (insulin-like growth restriction. Deletions or loss-of-function mutations in factor-2), and insulin are three key players. The expres- the IGF1 gene cause intrauterine and postnatal growth sion of IGF1 and IGF2 genes in fetal tissues and placenta retardation [4–7]. In mice, the deletion of the IGF2 gene is essential for fetal growth, as indicated by studies of reduces fetal growth . The effects on fetal growth are knock-out mice and genetic defects in human. Although additive to those of IGF1. IGF2 is an imprinted gene. IGF2 is more abundantly expressed in fetal serum and The expression of IGF2 maternal allele is silenced during tissues than IGF1, IGF1 is more closely associated with fetal life, leading to fetal growth restraint when the fetal growth in a majority of species. Insulin secreted by paternal allele is mutated or deleted. Epimutations and the fetal ß cells is also a major growth factor during molecular alterations of the human chromosomal region intrauterine life . 11p15.5, which harbors IGF2 , and paternally inher- ited nonsense mutations of the IGF2 gene  are associ- * Correspondence: email@example.com Institut National de la Santé et de la Recherche Médicale U1169, Bicêtre Hospital, Paris Sud University, Le Kremlin-Bicêtre, France Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Le Stunff et al. Clinical Epigenetics (2018) 10:57 Page 2 of 7 ated with the Silver–Russell syndrome, a syndromic population-based cohort . Another study of methyla- growth-retardation disorder. Heterozygous mutations in tion (performed on cord blood and placenta) restricted the INS gene are associated with a reduction of fetal to growth-related genes found no association between growth . intrauterine growth retardation and IUGR and the While monogenic defects in the IGF1, IGF2,and INS methylation of CpGs at the IGF1, IGF2, and INS loci genes demonstrate the major role of these genes for fetal (note that this study investigated only 3 CpGs located growth, the genetics of the individual variability in birth- within the IGF1 P1 promoter) . weight of normal neonates is complex, as for most quanti- The current study investigates the relationship be- tative traits, and relies on numerous polymorphic variations tween birth length and the methylation of specific CpGs of the genome. Birth size is a multifactorial phenotype of the IGF1 and INS genes. These CpGs located within resulting from many processes and gene expression pat- promoters were selected because their level of methyla- terns operating during fetal development. Genetic variation tion was known to control gene expression. This is the is said to account for 38–80% of birth weight variance, with case for IGF1 CpG-137, whose methylation is associated a considerable variability in the estimates [11, 12]. A with IGF1 gene transcription , circulating IGF1  genome-wide association study found 60 loci to be associ- and childhood growth [25, 26]. This is also the case for ated with birth weight at genome-wide significance . CpG-180 in the INS promoter region, which influences Despite their implication in monogenic disorders of fetal INS gene transcription  and is associated with SNPs growth, neither IGF1 nor INS loci were among these 60 as rs689  known to predict birth size to some degree loci, but pathways involved in insulin and IGF signaling . We have not explored a potential association of were said to be involved. As pointed out for most quantita- birth length with CpG methylation on the non- tive traits studied in humans, the genetics of birth size face imprinted allele of IGF2 because we did not have access the missing heritability problem. Indeed, in contrast with to parental genotypes. the estimates of birth weight heritability [12, 14], the 60 var- iants most recently identified could only explain 2% of the Methods variance in birth weight . When SNPs at the INS [15, Mothers 16]and IGF1 loci [17, 18] were examined specifically as One hundred and fifty-nine women of European ances- candidates for their contribution to the variation of size at try aged 20–40 years, with a singleton pregnancy, were birth, they showed inconsistent results [15, 19]. recruited at Antoine Béclère Maternity. The clinical The genotype of the fetus is not the sole determinant of characteristics of the 159 newborns are presented in fetal growth. It is likely that maternal genes that regulate Table 1. All were healthy neonates born after 37 weeks the environment of the womb are important determinants of amenorrhea. None had IUGR, defined as a weight or of birth size. Maternal environment also has a major influ- length below the 10th percentile for its gestational age ence, although most of the environmental factors and . Children born before 36 weeks of gestation were mechanisms of reduced fetal growth remain largely un- not included. Clinical data and biological samples were known. Non-inherited maternal factors found to influence collected at inclusion (< 3 months of pregnancy) and birth size include parity, mother weight at birth and before around delivery. The main characteristics of the mothers pregnancy, weight gain of pregnancy, and maternal smok- are presented in Table 1. All mothers filled a 142-item ing, but cannot explain the vast majority of cases of questionnaire. All mothers were healthy and had a nor- intrauterine growth retardation (IUGR) . Maternal nu- mal nutrition; none consumed alcohol or drugs during trition, in industrialized populations seems to have only a their pregnancy. All protocols were agreed by French small effect on placental and birth weights . Indeed, ethic boards (CODECOH DC-2013-2017, CPP CO-14- even in affluent societies where mothers are well nour- 001, CCTIRS no. 14-124bis, CNIL no. 914,253). Cord ished, many children are still born with unexplained small blood samples were taken within minutes of delivery, fetal size. On the other side, extreme maternal nutrition, immediately refrigerated at 4 °C and transported to la- such as the Dutch Hunger Winter famine, is associated boratory within 24 h. with a reduced birth weight . As for all quantitative traits, epigenetic influences are likely to contribute to the regulation of birth size at the Isolation of genomic DNA and bisulfite genomic individual level. Few studies have yet explored the epi- conversion genetics of fetal growth in humans. To our knowledge, Cord blood mononuclear cells (CBMC) were isolated the only epigenome-wide association study carried out from fresh blood using a density gradient. Five millime- yet found that among the 485,577 CpGs analyzed in ters of fresh blood was mixed with 5 ml of NaCl cord blood by the 450K Infinium BeadChip, 19 CpGs 154 mM, and 5 ml of Lymphoprep solution (Eurobio, were significantly associated with birthweight in a large Paris, France) was added to the diluted blood and Le Stunff et al. Clinical Epigenetics (2018) 10:57 Page 3 of 7 Table 1 Main characteristics of the studied newborns and designed using MethPrimer (htpp://urogene.org/meth- mothers (mean ± sd) primer/index1.html). Biotin-labeled single N 159 stranded amplicon was isolated according to protocol using the Qiagen Pyromark Q96 Work Station and Sex (M/F) 84/75 underwent pyrosequencing with 0.5 μMofsequencing Term of birth (weeks) 39.8 ± 1.1 primer. The methylation percent for each CpG within Birth length (cm) 49.4 ± 1.4 the target sequence was calculated using PyroQ CpG Birth length (SDS) − 0.43 ± 0.6 Software (Qiagen). Birth weight (g) 3355 ± 330 Birth weight (SDS) 0.19 ± 0.7 Genotyping of SNPs at the IGF1 and INS loci Midparental height (cm) 169 ± 7 Genotyping of SNP rs35767 at the IGF1 gene locus was IGF1 (ng/ml) 50 ± 19 performed with TaqMan allelic discrimination using Bio- Insulin (μU/ml) 4.5 ± 4 systems 7500 Fast Real-Time PCR System (Applied Bio- Mother’s age (years) 32.1 ± 3.9 systems, Courtaboeuf, France). SNP genotyping assay Mother weigh gain (kg) 10.9 ± 4 (ID: C_7999146_10) was purchased from Life technolo- Smoking (%) 6 gies (Saint Aubin, France). Genotyping of SNP rs689 at the INS gene locus was Primaparous (%) 35 determined by the analysis of PCR products . PCR Folate supplement before conception (%) 32 amplification was in 96-well microliter plates, each 50 μl Folate supplement during pregnancy (%) 79 reaction contained DNA (100 ng), MgCl (1.5 mM), 10× reaction Buffer (Thermo Scientific), dNTPs (2.0 mM each), primers (1 μM each), and Taq Polymerase (1. centrifuged for 20 min at room temperature at 800 g. 25 U, Thermo Scientific, Saint Aubin, France). Amplified After centrifugation, the interphase containing CBMC PCR products were digested with 1 unit of Hph1 en- was carefully aspirated and the cells were mixed with zyme (Thermo Scientific, Saint Aubin, France). NaCl. The cell suspension was centrifuged at 300 g, and For genotyping quality, negative controls were in- the cell pellet washed with PBS. cluded in each PCR plate. Twenty percent of samples Nucleic acids were extracted from CBMC using Gen- were analyzed as duplicate for genotyping determination. tra Puregene blood kit (Qiagen, Hilden, Germany). Four The Hardy-Weinberg equilibrium (HWE) was calculated hundred nanograms of genomic DNA was treated with by computing the test for deviations in HWE and was EZ DNA Methylation-Gold Kit, according to the manu- shown to be present across genotypes. Allele frequencies facturer’s protocol (Zymo Research Corporation, CA, were calculated and tested by test. USA). A bisulfite-PCR-pyrosequencing technique was used to measure the CG methylation . We improved the resolution of this method from a handful of bases to Measurement of serum IGF1 and insulin concentrations up to 100 nucleotides, with the ability to quantify IGF1 and insulin concentrations were measured in methylation in the same sample of blood cells with a co- serum samples from the cord blood of newborns. efficient of variation (sd/mean) as little as 1–5% [25, 28]. IGF1 concentration was measured using a chemilu- minescent immunometric assay after pre-treatment Pyrosequencing-based bisulfite PCR analysis with acid using Immulite®2000 (Siemens Healthcare CpGs are denominated according to their position Diagnostics Products Llanberis, UK). Insulin concen- versus each promoter transcription start site (TSS). At tration was measured using Liaison®Insulin (DiaSorin, the IGF1 locus, we studied 3 CpGs (-1044, -960, -919) Saluggia, Italy). located within the P1 promoter and 5 CpGs (-232, -224, -218, -207, -137) located within the P2 promoter (Additional file 1: Figure S1). We previously have shown that the methylation of the P1 promoter does not influ- Statistical analysis ence IGF1 gene expression . At the INS locus, we Birth weight (sds) and birth length (sds) were regressed on studied 2 CpGs (-206 and -180) proximal to the TSS methylation of CpG-137, sex, folate, supplement intake (Additional file 1: Figure S1). The bisulfite-treated gen- before conception (yes/no), folate supplement intake dur- omic DNA was PCR-amplified using unbiased primers ing pregnancy (yes/no), parity (primiparous/non primipar- (Additional file 2: Table S1) and performed quantitative ous), maternal age, and smoking (yes/no) in multivariate pyrosequencing using a PyroMark Q96 ID Pyrosequenc- linear regression. Results are expressed as mean ± sd. All ing instrument (Qiagen). Pyrosequencing assay was statistical analyses were conducted using R 3.3.2. Le Stunff et al. Clinical Epigenetics (2018) 10:57 Page 4 of 7 Results Effects of smoking and folate on birth weight Six percent of the newborns were from smoking mothers. The mean birth length of neonates born to a smoking mother was 0.62 SDS (95% CI 0.22–1.04) lower than that of neonates born to a non-smoking mother (P −3 = 3.0 × 10 ), a result retrieved in the multivariate −3 analysis (P = 5.6 × 10 ). Thirty-two percent of women took a folate supplement before conception. The bivari- ate analysis showed a trend for increased birth weight associated with folate intake before conception (P = 0.09) , an association found strengthened in the multivariate −3 regression (P = 9.2 × 10 ). No association between birth length or birth weight was found with a supplement of folate during pregnancy. Neither smoking nor folate intake were associated with variations in CpG-137 methylation. Epigenetic and genetic variation at the IGF1 locus While methylation of CpG-218 located within the IGF1 Fig. 1 CpG-137 methylation correlates negatively with birth length. The P2 promoter correlated closely with methylation of CpG- −15 correlation between CpG-137 (%) and birth length (SDS) is described by 137 (P =9.4 × 10 ), methylation of CpGs -1044, -960, the equation: Birth length = − 0.023 × [CpG-137 methylation] + 0.68 with and -919, located within the P1 promoter (Table 2), −4 r =0.2 and P =3.5 ×10 showed no correlation with CpG-137 methylation (Additional file 3:FigureS2).Wefound no correl- ation between the rs35767 genotype and the methyla- Cord blood IGF1 concentration showed no association with tion of CpG-137 of the P2 or other studied CpGs methylation at CpG-137 (r =0.06, P =0.61). (Additional file 4:FigureS3A). Methylation at CpG-224 and CpG-218 correlated Birth length correlated negatively with methylation at negatively with birth length (P = 0.03 and P = 0.02, −4 CpG-137 (r =0.2, P =3.5× 10 ,Fig. 1). This finding was respectively) (Additional file 5: Figure S4). confirmed after adjustment for several covariates: a 10% increase in methylation at CpG-137 was associated with a Epigenetic and genetic variation at the INS locus decrease of birth length by 0.23 SDS (95% CI 0.11–0.35; Methylation of INS CpG-180 and CpG-206 showed no −4 P =1.6 ×10 ). In contrast, methylation at CpG-137 was not correlation with birth length or birth weight. Birth associated with birth weight (P =0.36). The same result was length and birth weight were not associated with rs689 obtained in the multivariate regression framework (P =0.15). alleles (Additional file 6: Figure S5). As previously reported , we found that CpG-206 Table 2 CpG methylation (%) in the promoters of the IGF1 and and CpG-180 methylation in the studied newborns was INS genes of the studied newborns strongly influenced by rs689 alleles (Additional file 4: IGF1 gene Promoter 1 −16 Figure S3B; P < 2.10 ). CpG-1044 89 ± 4% CpG-960 82 ± 3% Discussion The fetal genotype explains only a limited part of intra- CpG-919 93 ± 9% uterine growth variability among individuals of the gen- IGF1 gene Promoter 2 eral population , which leaves an important role to CpG-232 57 ± 8% maternal genotype, maternal environment, and fetal and CpG-224 67 ± 9% placental epigenetics. Animal studies have demonstrated CpG-218 68 ± 9% that environment can shape the epigenome, notably dur- CpG-207 43 ± 6% ing the intrauterine period when it has the greatest plas- ticity. Epigenetic effects of the intrauterine environment CpG-137 49 ± 8% can thus influence the phenotype in later life. However, INS gene Promoter it remains unclear as to how influential the fetal period CpG-180 61 ± 8% is in shaping the epigenome, whether different genomic CpG-206 83 ± 5% regions show varying sensitivities to this environment Le Stunff et al. Clinical Epigenetics (2018) 10:57 Page 5 of 7 during this period, and the extent to which this early Several hypotheses may account for the association interaction is sensitive to genetic influences. of methylation at CpG-137 measured at birth with To our knowledge, few studies have examined the as- fetal growth. A first possibility is that the individual sociation of CpG methylation with fetal growth. The first variation in CpG-137 methylation is determined in study found an increased methylation of all CpG sites at the post implantatory embryo, at time of the primary the IGF1 P1 promoter in the placenta of fetuses with shaping of the methylome. Alternatively, the vari- IUGR , associated with the previously reported de- ation observed in the level of methylation of CpG- creased IGF1 gene expression . Another study used 137 may result from yet unknown maternal signals the Illumina Infinium HM27 platform to profile CpG transmitted to the fetal tissues through the placenta methylation in CBMC in a small number of twin pairs at a post-embryonic stage of intrauterine life. No as- . Array technology, however, uses CpGs that are not sociation of fetal growth was observed with the necessarily those having specific effects on gene expres- other CpGs of the IGF1 locus, except for CpG-218, sion and phenotypes. This was the case at the IGF1 another CpG of the P2 promoter. locus for the array used in twins, which analyzed only 1 Maternal smoking  or folate intake has CpG of the P1 promoter and no CpG of the P2 pro- been shown in other studies to affect methylation of moter at the IGF1 locus, while it examined 4 CpGs at certain CpGs, but was not found to affect the the INS locus. Findings were that none of these CpGs methylation of CpG-137 or of other CpGs studied had their methylation level associated with twins’ birth- herein. weight. A third study of methylation restricted to Since insulin is a major growth factor in fetal life growth-related genes found no association between and the INS VNTR has a direct effect on insulin IUGR and the methylation of CpGs at the IGF1, IGF2, transcription , it is conceivable that INS VNTR and INS loci in placenta or cord blood samples . variation influences early growth. This is why CpG- Instead, our study focused on CpGs located within the 180 and CpG-206 located within the INS promoter regulatory region of IGF1 and INS genes, because these were selected for the current study, given that their two genes are known to be major contributors to fetal level of methylation affects the expression of the in- growth. Some of these CpGs were selected because their sulin gene [27, 28]. As reported previously the methylation was already known to be associated with gene methylation of CpG-206 and CpG-180 was strongly expression [25, 28], the others because they were located influenced by rs689 alleles. We found that neither within neighboring regulatory regions. We found that rs689 alleles nor CpG-206 or CpG-180 showed a re- CpG-137 methylation was negatively associated with birth lationship with birth length or birth weight, but this length in normal neonates. The methylation of CpG-137 conclusion should await the observation of a large has a strong functional role upon IGF1 gene expression in number of neonates. Unlike Dunger et al. , we children’sPBMCand in theHEK293cellline. It was observed no association (Additional file 4:FigureS3) previously shown to be associated with postnatal growth between birth size and insulin VNTR classes or variability . If methylation at CpG-137 in CBMC is a rs689 (in complete linkage disequilibrium with proxy of the methylation in growth-promoting tissues of VNTR classes). This may be due to the fact that we the fetus (which has not been shown in the current study), were not able to distinguish “non-changers”  one can speculate that methylation at this CpG influences among the studied neonates: Non-changers is the IGF1 gene expression and IGF1 production in fetal tissues, term used to describe infants that do not show a thus fetal growth. There is no evidence, however, that the catch-up growth after being born small for gesta- individual variation of CpG methylation reflects that in tional age. growth-promoting tissues. While this is a major weakness of our study, this weakness is shared with a majority of studies on epigenetics in humans where blood cells are Conclusions used as proxies of physiological tissues, given that clinical In conclusion, fetal growth in normal neonates is research does not have access to these tissues . Another associated with the methylation of CpG-137 located weakness of measuring CpG methylation in CBMC is that in the IGF1 P2 promoter. This observation supports it is a unique cell mixture containing red blood cells in a significant role of IGF1 epigenetics in the regula- addition to other blood cells, and not a well-characterized tion of fetal growth that does not seem to be homogeneous cell type. Our study was not able to estimate dependent on cis-genetic variation at the IGF1 cell composition of this mixture based on methods devel- locus. Although small, this epigenetic effect is of an oped for adult blood cells , nor to sort a specific cell order of magnitude comparable with that of many category from the cord blood sample. genomic variants associated with human quantita- tive traits. Le Stunff et al. Clinical Epigenetics (2018) 10:57 Page 6 of 7 Additional files Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional file 1: Figure S1. Schematic representation of the IGF1 and INS loci. (A) The two IGF1 gene promoters are figured. CpGs are indicated Author details as lollypops (studied CpGs in white and non-studied CpG in black). Institut National de la Santé et de la Recherche Médicale U1169, Bicêtre rs35767 is indicated by a black arrow. TSS are shown as broken arrows. Hospital, Paris Sud University, Le Kremlin-Bicêtre, France. Service de (B) INS promoter is figured. CpGs are indicated as lollypops (studied CpGs Médecine des Adolescents, Bicêtre Hospital, Paris Sud University, Le in white and non-studied CpG in black). TSS is shown as broken arrow. Kremlin-Bicêtre, France. Service de BiologieMoléculaire et Hormonologie, Location of primers are indicated by arrow for CpG methylation and Bicêtre Hospital, Paris Sud University, Le Kremlin-Bicêtre, France. Service de genotyping. Sequences of primers and location on chromosome are Gynécologie-Obstétrique, Antoine Béclère Hospital, Paris Sud University, provided in Additional file 2: Table S1. (PPTX 67 kb) Clamart, France. CRCINA, INSERM U1232, Université de Nantes, Nantes, Additional file 2: Table S1. List of primers and location used in our France. study. Sequences are given from 5′ to 3′. (DOCX 15 kb) Received: 17 October 2017 Accepted: 4 April 2018 Additional file 3: Figure S2. Correlation matrix of methylation values (%) at the CpG located in the P1 and P2 promoters of the IGF1 gene in newborns patients. Pearson correlation coefficient is indicated in bold, and P value below. (PPTX 88 kb) References Additional file 4: Figure S3. Relationship between promoter CpG 1. Gatford KL, Simmons RA. Prenatal programming of insulin secretion in methylation and genotypes. (A) Methylation at CpGs-137 of the IGF1 P2 intrauterine growth restriction. Clin Obstet Gynecol. 2013;56(3):520–8. promoter is independent from the rs35767 genotypes. (B) Methylation at 2. Baker J, Liu JP, Robertson EJ, Efstratiadis A. Role of insulin-like growth factors CpGs-206 and CpG-180 in insulin promoter is closely dependent on rs689 in embryonic and postnatal growth. Cell. 1993;75(1):73–82. alleles. (PPTX 242 kb) 3. Fowden AL. The insulin-like growth factors and feto-placental growth. Additional file 5: Figure S4. Relation between CpG methylation and Placenta. 2003;24(8–9):803–12. birth length (SDS) at the IGF1 promoter 1 and 2. (A) at IGF1 P1 promoter, 4. Abuzzahab MJ, Schneider A, Goddard A, Grigorescu F, Lautier C, Keller E, we observed no significant correlation of birth length with the studied Kiess W, Klammt J, Kratzsch J, Osgood D, et al. IGF-I receptor mutations CpGs, (B) at IGF1 P2 promoter, only two CpGs other that CpG-137 resulting in intrauterine and postnatal growth retardation. N Engl J Med. showed a weak correlation with birth length. The correlation between 2003;349(23):2211–22. CpG-224 (%) and birth length (SDS) is described by the equation: Birth 5. Woods KA, Camacho-Hubner C, Bergman RN, Barter D, Clark AJ, Savage MO. length = − 0.014*[CpG-224 methylation] + 0.43 (r = 0.17, P = 0.03). The Effects of insulin-like growth factor I (IGF-I) therapy on body composition correlation between CpG-218 (%) and birth length (SDS) is described by and insulin resistance in IGF-I gene deletion. J Clin Endocrinol Metab. 2000; the equation: Birth length = − 0.016*[CpG-218 methylation] + 0.58 (r =0.2, 85(4):1407–11. P = 0.02). (PPTX 752 kb) 6. Bonapace G, Concolino D, Formicola S, Strisciuglio P. A novel mutation in a Additional file 6: Figure S5. Relationship between insulin rs689 genotype patient with insulin-like growth factor 1 (IGF1) deficiency. J Med Genet. and birth weight (sds) and birth length (sds). Birth weight (sds) and birth 2003;40(12):913–7. length (sds) are independent from the rs 689 genotypes. (PPTX 161 kb) 7. Netchine I, Azzi S, Houang M, Seurin D, Perin L, Ricort JM, Daubas C, Legay C, Mester J, Herich R, et al. 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Published: Apr 19, 2018