Grandmaternal smoking during pregnancy is associated with differential DNA methylation in peripheral blood of their grandchildren.

The idea that information can be transmitted to subsequent generation(s) by epigenetic means has been studied for decades but remains controversial in humans. Epidemiological studies have established that grandparental exposures are associated with health outcomes in their grandchildren, often with sex-specific effects; however, the mechanism of transmission is still unclear. We conducted Epigenome Wide Association Studies (EWAS) to test whether grandmaternal smoking during pregnancy is associated with altered DNA methylation (DNAm) in peripheral blood from their adolescent grandchildren. We used data from a birth cohort, with discovery and replication datasets of up to 1225 and 708 individuals (respectively, for the maternal line), aged 15-17 years, and tested replication in the same individuals at birth and 7 years. We show for the first time that DNAm at a small number of loci in cord blood is associated with grandmaternal smoking in humans. In adolescents we see suggestive associations in regions of the genome which we hypothesised a priori could be involved in transgenerational transmission - we observe sex-specific associations at two sites on the X chromosome and one in an imprinting control region. All are within transcription factor binding sites (TFBSs), and we observe enrichment for TFBS among the CpG sites with the strongest associations; however, there is limited evidence that the associations we see replicate between timepoints. The implication of this work is that effects of smoking during pregnancy may induce DNAm changes in later generations and that these changes are often sex-specific, in line with epidemiological associations.

Paternal grandmother's smoking in pregnancy is associated with extreme aversion to bitter taste in their grandchildren.

Although there are many examples in the experimental literature of an environmental exposure in one generation impacting the phenotypes of subsequent generations, there are few studies that can assess whether such associations occur in humans. The Avon Longitudinal Study of Parents and Children (ALSPAC) has, however, been able to determine whether there are associations between grandparental exposures and their grandchildren's development. Several of our studies, including sensitivity to loud noise, have shown associations between a grandmother smoking in pregnancy and the phenotype of the grandchild. These results were mostly specific to the sex of the grandchild and to whether the prenatal (i.e. during pregnancy) smoking occurred in the maternal or paternal grandmother. Here, we have used ancestral data on prenatal smoking among the grandmothers of the ALSPAC index children to examine possible effects on the grandchild's ability to detect the bitter taste of PROP (6 n-propylthiouracil), distinguishing between the 10% deemed 'extreme tasters', and the rest of the population (total N = 4656 children). We showed that grandchildren whose paternal (but not maternal) grandmothers had smoked in pregnancy were more likely than those of non-smoking grandmothers to be extreme tasters [odds ratio (OR) 1.28; 95% confidence interval (CI) 1.03, 1.59] and that this was more likely in granddaughters (OR 1.42; 95% CI 1.03, 1.95) than grandsons (OR 1.18; 95% CI 0.88, 1.60). This pattern of association between paternal foetal exposure and the granddaughter's development has been found with several other outcomes, suggesting that investigations should be undertaken to investigate possible mechanisms.

Human transgenerational observations of regular smoking before puberty on fat mass in grandchildren and great-grandchildren.

Previously, using data from the Avon Longitudinal Study of Parents and Children (ALSPAC) we showed that sons of fathers who had started smoking regularly before puberty (< 13 years) had increased fat mass during childhood, adolescence, and early adulthood. We now show that if the paternal grandfather had started smoking pre-puberty, compared with later in childhood (13-16 years), his granddaughters, but not grandsons, had evidence of excess fat mass at two ages: mean difference + 3.54 kg; (P with 1-tailed test) = 0.043 at 17 years, and + 5.49 kg; (P1 = 0.016) at age 24. When fathers of maternal grandfathers had started smoking pre-puberty, their great-granddaughters, but not great-grandsons, had excess body fat: + 5.35 kg (P1 = 0.050) at 17, and + 6.10 kg (P1 = 0.053) at 24 years. Similar associations were not found with lean mass, in a sensitivity analysis. To determine whether these results were due to the later generations starting to smoke pre-puberty, further analyses omitted those in subsequent generations who had smoked regularly from < 13 years. The results were similar. If these associations are confirmed in another dataset or using biomarkers, this will be one of the first human demonstrations of transgenerational effects of an environmental exposure across four generations.

Regular smoking of male ancestors in adolescence and fat mass in young adult grandchildren and great-grandchildren.

Background: Previous studies using the Avon Longitudinal Study of Parents and Children (ALSPAC) have shown that if men commenced smoking prior to the onset of puberty their sons, their granddaughters and great-granddaughters were more likely to have excess fat (but not lean) mass during childhood, adolescence and early adulthood. In this study we assess associations between ancestral smoking during adolescence (ages 11-16 years) with fat and lean mass of subsequent generations at two ages. Methods: We analysed data on exposures of grandparents and great-grandparents collected by ALSPAC. The outcomes were the fat masses of their grandchildren and great-grandchildren measured at ages 17 and 24. Measures of lean mass were used as controls. Adjustment was made for 8-10 demographic factors using multiple regression. Results: We found associations between adolescent smoking of the paternal grandfathers and the adjusted fat mass of their grandchildren, but no associations with the grandchildren's lean mass. Grandchildren at age 17 had an average excess fat mass of +1.65 [95% CI +0.04, +3.26] Kg, and at age 24 an average excess of +1.55 [95% CI -0.27, +3.38] Kg. Adolescent smoking by the maternal grandfather showed similar, but weaker, associations: at 17 an average excess fat mass of +1.02 Kg [95% CI -0.20, +2.25] Kg, and at 24 an average excess of +1.28 [95% CI -0.11, +2.66] Kg. There were no pronounced differences between the sexes of the children. For the great-grandparents there were few convincing results, although numbers were small. Conclusions: We have shown associations between grandfathers' smoking in adolescence and increased fat (but not lean) mass in their children. Confirmation of these associations is required, either in a further data set or by demonstrating the presence of supportive biomarkers.

Ancestral smoking and developmental outcomes: a review of publications from a population birth cohort†.

The adverse effects on the child of maternal smoking in pregnancy is well-recognized, but little research has been carried out on the possible non-genetic effects of ancestral smoking prior to the pregnancy including parental initiation of cigarette smoking in their own childhoods or a grandmother smoking during pregnancy. Here, we summarize the studies that have been published mainly using data from the Avon Longitudinal Study of Parents and Children. We demonstrate evidence that ancestral smoking prior to or during pregnancy can often be beneficial for offspring health and both ancestor- and sex-specific. More specifically, we report evidence of (i) adverse effects of the father starting to smoke pre-puberty on his son's development; (ii) beneficial effects on the grandson if his maternal grandmother had smoked in pregnancy; and (iii) mainly adverse effects on the granddaughter when the paternal grandmother had smoked in pregnancy. The ancestor- and sex-specificity of these results are consistent with earlier studies reporting associations of health and mortality with ancestral food supply in their parents' and grandparents' pre-pubertal childhoods.

Unexpected Associations between the Number of FRAXE Repeats in Boys and Evidence of Diabetes in Their Mothers and Maternal Grandmothers.

The FRAXE section of the FMR2 gene, located on the X chromosome, contains varying numbers of trinucleotide repeats; boys with over 200 repeats tend to have mild cognitive impairments, though this is rare. Little is known, however, concerning the phenotypes of individuals with smaller numbers of repeats. Here we answer the research question as to whether the health of ancestors of boys from whom the relevant X chromosome was inherited differed in any way according to the number of FRAXE repeats. Numbers of FRAXE repeats in 5057 boys from the Avon Longitudinal Study of Parents and Children (ALSPAC) were assessed. The distribution was bimodal, with the second smaller distribution starting at 22 repeats. We tested whether possession of 22+ repeats was associated with differences in the health of mothers (who share the X chromosome) and maternal grandmothers (half of whom share it). Female ancestors of boys with >21 repeats compared with <22 showed that maternal grandmothers (MGM) and mothers (M) had an increased risk of diabetes: MGM Type I odds ratio (OR) 2.40 [95%CI: 1.07,5.38]; MGM Type II OR 1.61 [0.96,2.70]; M OR 1.95 [0.96,3.94] using self-reported questionnaire measures. These results were confirmed from maternal medical records which revealed an increased level of diabetes [OR 2.40 (1.16,4.96)] and an increased risk of repeated glycosuria during pregnancy [OR 1.60 (1.08,2.36)]. We tested numbers of FRAXA repeats and showed no such associations, indicating that the findings were not associated with triploid repeats in general. If these findings are replicated elsewhere, there are at least three possible interpretations: (i) maternal diabetes/prediabetes results in an increased number of FRAXE repeats; (ii) women with high numbers of FRAXE repeats are at increased risk of diabetes; or (iii) some common factor, e.g. genomic instability, results in both diabetes and increased repeats.

Ancestral childhood environmental exposures occurring to the grandparents and great-grandparents of the ALSPAC study children.

Background: Cohort studies tend to be designed to look forward from the time of enrolment of the participants, but there is considerable evidence that the previous generations have a particular relevance not only in the genes that they have passed on, their cultural beliefs and attitudes, but also in the ways in which previous environmental exposures may have had non-genetic impacts, particularly for exposures during fetal life or in childhood. Methods: To investigate such non-genetic inheritance, we have collected information on the childhoods of the ancestors of the cohort of births comprising the original Avon Longitudinal Study of Parents and Children (ALSPAC). The data collected on the study child's grandparents and great grandparents comprise: (a) countries of birth; (b) years of birth; (c) age at onset of smoking; (d) whether the ancestral mothers smoked during pregnancy; (e) social class of the household; (f) information on 19 potentially traumatic situations in their childhoods such as death of a parent, being taken into care, not having enough to eat, or being in a war situation; (g) causes of death for those ancestors who had died. The ages at which the individual experienced the traumatic situations distinguished between ages <6; 6-11, and 12-16 years. The numbers of ancestors on which data were obtained varied from 1128 paternal great-grandfathers to 4122 maternal great grandmothers. These ancestral data will be available for analysis to bona fide researchers on application to the ALSPAC Executive Committee.

Intolerance of loud sounds in childhood: Is there an intergenerational association with grandmaternal smoking in pregnancy?

Recent research using the Avon Longitudinal Study of Parents and Children (ALSPAC) demonstrated an association between maternal grandmother smoking in pregnancy and the autistic traits of impaired social communication and repetitive behaviour in granddaughters but not grandsons, but of paternal grandmother smoking and early development of myopia in the grandchild. Here we investigate whether grandmaternal smoking in pregnancy is associated with intolerance to loud sounds. ALSPAC collected information during the index pregnancy from the study parents on the smoking habits, social and other features of their own parents. Maternal report when the child was aged 6 and 13 included hating loud sounds; at age 11 the child was tested for volume preference for listening to music through headphones. Statistical analysis compared results for grandchildren in relation to whether a parent had been exposed in utero to maternal smoking, adjusted for their grandparents' social and demographic attributes. We hypothesised that there would be sex differences in the effects of grandmaternal prenatal smoking, based on previous intergenerational studies. For 6-year-old children maternal report of intolerance to loud noise was more likely in grandsons if the maternal grandmother had smoked [adjusted odds ratio (AOR) 1.27; 95% confidence interval (CI) 1.03,1.56; P = 0.025], but less likely in girls [AOR 0.82; 95%CI 0.63,1.07] Pinteraction <0.05. If the paternal grandmother had smoked the grandchildren were less likely to be intolerant, especially girls. The objective measure of choice of volume for music through headphones showed that grandsons of both maternal and paternal smoking grandmothers were less likely to choose high volumes compared with granddaughters (P<0.05). In line with our prior hypothesis of sex differences, we showed that grandsons were more intolerant of loud sounds than granddaughters particularly at age 6, and this was confirmed by objective measures at age 11.

Grandchild's IQ is associated with grandparental environments prior to the birth of the parents.

Background. Despite convincing animal experiments demonstrating the potential for environmental exposures in one generation to have demonstrable effects generations later, there have been few relevant human studies. Those that have been undertaken have demonstrated associations, for example, between exposures such as nutrition and cigarette smoking in the grandparental generation and outcomes in grandchildren. We hypothesised that such transgenerational associations might be associated with the IQ of the grandchild, and that it would be likely that there would be differences in results between the sexes of the grandparents, parents, and children. Method. We used three-generational data from the Avon Longitudinal Study of Parents and Children (ALSPAC).  We incorporated environmental factors concerning grandparents (F0) and focussed on three exposures that we hypothesised may have independent transgenerational associations with the IQ of the grandchildren (F2): (i) UK Gross Domestic Product (GDP) at grandparental birth year; (ii) whether grandfather smoked; and (iii) whether the grandmother smoked in the relevant pregnancy. Potential confounders were ages of grandparents when the relevant parent was born, ethnic background, education level and social class of each grandparent. Results. After adjustment, all three target exposures had specific associations with measures of IQ in the grandchild. Paternal grandfather smoking was associated with reduced total IQ at 15 years; maternal grandfather smoking with reduced performance IQ at 8 years and reduced total IQ at 15.  Paternal grandmother smoking in pregnancy was associated with reduced performance IQ at 8, especially in grandsons. GDP at grandparents' birth produced independent associations of reduced IQ with higher GDP; this was particularly true of paternal grandmothers. Conclusions. These results are complex and need to be tested in other datasets. They highlight the need to consider possible transgenerational associations in studying developmental variation in populations.

Author Correction: Grandmothers' smoking in pregnancy is associated with a reduced prevalence of early-onset myopia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Grandmothers' smoking in pregnancy is associated with a reduced prevalence of early-onset myopia.

Myopia (near sightedness) is the most common vision disorder resulting in visual impairment worldwide. We tested the hypothesis that intergenerational, non-genetic heritable effects influence refractive development, using grandparental prenatal smoking as a candidate exposure. Using data from the Avon Longitudinal Study of Parents and Children (ALSPAC), we found that the prevalence of myopia at age 7 was lower if the paternal grandmother had smoked in pregnancy, an association primarily found among grandsons compared to granddaughters. There was a weaker, non-sex-specific, reduction in the prevalence of myopia at age 7 if the maternal grandmother had smoked in pregnancy. For children who became myopic later (between 7 and 15 years of age) there were no associations with either grandmother smoking. Differences between early and late-onset myopia were confirmed with DNA methylation patterns: there were very distinct and strong associations with methylation for early-onset but not later-onset myopia.

Investigating Possible Trans/Intergenerational Associations With Obesity in Young Adults Using an Exposome Approach.

Animal experiments demonstrate ways in which an exposure in one generation can be reflected in a variety of outcomes in later generations. In parallel human observational studies have shown associations between grandparental and parental exposures to cigarette smoking and/or nutrition and growth and survival of the grandchild. These studies have controlled for just a few confounders selected ad hoc. Here we use an exposome approach (using all available measures of exposure) to determine trans/inter-generational factors that may be important in studying environmental factors associated with fat mass in young human adults. The study takes advantage of the rich data available in the Avon Longitudinal Study of Parents and Children (ALSPAC). We test associations with features of grandparents (G0) and the childhood of the parents (G1) of 24-year olds (G2). We hypothesized that intergenerational associations would be revealed, particularly with exposure to cigarette smoke, and that these would vary with the sexes of all three generations. The study exposome analyzed 172 exposures to the maternal line and 182 to the paternal line. A series of stepwise regression analyses reduced the initial 40 unadjusted factors (P < 0.05) to eight independent features on the maternal line, and of 26 on the paternal line to five. We found strong associations between the father starting to smoke cigarettes regularly before age 11 and increased fat mass in his adult children (unadjusted = +7.82 [95% CI +2.75, +12.90] Kg; adjusted = +11.22 [+5.23, +17.22] Kg); this association was stronger in male offspring. In addition, when the paternal grandmother had smoked in pregnancy her adult granddaughters, but not grandsons had elevated mean fat mass (interaction with sex after adjustment, P = 0.001). The exposome technique identified other factors that were independently associated with fat mass in young adults. These may be useful in identifying appropriate confounders in other more proximal analyses, but also may identify features that may be on epigenetic pathways leading to increased fat mass in subsequent generations. We acknowledge that the results need to be replicated in other cohorts and encourage further linkage of outcomes with previous generational exposures, particularly along the paternal line.

The FRAXA and FRAXE allele repeat size of boys from the Avon Longitudinal Study of Parents and Children (ALSPAC).

The FRAXA and FRAXE alleles of the FMR1 and FMR2 genes located on the X chromosome contain varying numbers of trinucleotide repeats. Large numbers of repeats at FRAXA (full mutations) manifest as Fragile X syndrome, associated with mental impairment that affects males more severely. In this paper, we present the dataset of frequencies of FRAXA and FRAXE repeat size extracted from DNA samples collected from boys enrolled in the Avon Longitudinal Study of Parents and Children (ALSPAC). DNA data were extracted from samples collected in ALSPAC clinics from several types of samples: cord blood, venepuncture blood taken at 43 months, 61 months, seven years or nine years. The DNA was amplified at FRAXA and FRAXE using fluorescent PCR in the Wessex Regional Genetics Laboratory, Salisbury District Hospital. The mean repeat size for FRAXA is 28.92 (S.D. 5.44), the median 30 and the range 8 to 68. There were particularly high numbers of boys with repeat sizes of 20 (10.67%) and 23 (7.35%). The mean repeat size for FRAXE is 17.41 (S.D. 3.94), with median of 16 and range of 0 to 61. There is a relatively high degree of variation of the FRAXA repeat size particularly and we suggest the extensive data available from the ALSPAC study opens up areas of research into understanding phenotypes associated with relatively unexplored repeat sizes. This could be particularly interesting for the lower repeat sizes occurring with high frequency at FRAXA in this population. As the data can be linked to exposures and phenotypes, it will provide a resource for researchers worldwide.

Does cross-generational epigenetic inheritance contribute to cultural continuity?

Human studies of cross-generational epigenetic inheritance have to consider confounding by social patterning down the generations, often referred to as 'cultural inheritance'. This raises the question to what extent is 'cultural inheritance' itself epigenetically mediated rather than just learnt. Human studies of non-genetic inheritance have demonstrated that, beyond foetal life, experiences occurring in mid-childhood before puberty are the most likely to be associated with cross-generational responses in the next generation(s). It is proposed that cultural continuity is played out along the axis, or 'payoff', between responsiveness and stability. During the formative years of childhood a stable family and/or home permits small children to explore and thereby learn. To counter disruptions to this family home ideal, cultural institutions such as local schools, religious centres and market places emerged to provide ongoing stability, holding the received wisdom of the past in an accessible state. This cultural support allows the growing child to freely indulge their responsiveness. Some of these prepubertal experiences induce epigenetic responses that also transfer molecular signals to the gametes through which they contribute to the conception of future offspring. In parallel co-evolution with growing cultural support for increasing responsiveness, 'runaway' responsiveness is countered by the positive selection of genetic variants that dampen responsiveness. Testing these ideas within longitudinal multigenerational cohorts will need information on ancestors/parents' own communities and experiences (Exposome scans) linked to ongoing Phenome scans on grandchildren; coupled with epigenome analysis, metastable epialleles and DNA methylation age. Interactions with genetic variants affecting responsiveness should help inform the broad hypothesis.

Grand-maternal smoking in pregnancy and grandchild's autistic traits and diagnosed autism.

Although there is considerable research into the genetic background of autism spectrum disorders, environmental factors are likely to contribute to the variation in prevalence over time. Rodent experiments indicate that environmental exposures can have effects on subsequent generations, and human studies indicate that parental prenatal exposures may play a part in developmental variation. Here we use the Avon Longitudinal Study of Parents and Children (ALSPAC) to test the hypothesis that if the mother or father (F1) had been exposed to their own mother's (F0) smoking during pregnancy, the offspring (F2) would be at increased risk of autism. We find an association between maternal grandmother smoking in pregnancy and grand daughters having adverse scores in Social Communication and Repetitive Behaviour measures that are independently predictive of diagnosed autism. In line with this, we show an association with actual diagnosis of autism in her grandchildren. Paternal grandmothers smoking in pregnancy showed no associations.

Lymphoblastoid cell lines reveal associations of adult DNA methylation with childhood and current adversity that are distinct from whole blood associations.

BACKGROUND: Some cohort studies bank lymphoblastoid cell lines (LCLs) as a renewable source of participant DNA. However, although LCL DNA has proved valuable for genetic studies, its utility in epigenetic epidemiology research is unknown. METHODS: To assess whether LCL DNA can be used for life-course environmental epigenomics, we carried out a pilot methylomic study (using the Illumina Infinium Human Methylation 450 BeadChip) of nil-passage, Epstein-Barr virus (EBV)-transformed LCLs (n = 42) and 28 matched whole-blood (WB) samples. These were from adult male participants of the British 1958 birth cohort, selected for extremes of social economic position (SEP) in childhood and adulthood, with additional information available on childhood abuse and prenatal tobacco exposure. RESULTS: We identified a small number of weak associations between these exposures and methylation levels of both individual CpG sites and genomic regions in WB and LCLs. However, only one of the regional, and none of the individual CpG site associations were common to both sample types. The lack of overlap between the associations detected in LCL compared with those found in WB could either be due to the EBV-transformation process, or to the fact that, unlike WB, LCLs are essentially a single (CD19+) cell type. We provide evidence that the latter is the more potent explanation, by showing that CpG sites known to be differentially methylated between different types of blood cell have significantly lower correlations (R = 0.11) than average (R = 0.2) between WB and LCLs in our datasets, whereas sites known to be affected by EBV-transformation have significantly higher correlations (R = 0.3). CONCLUSIONS: This small pilot study suggests that the DNA methylation profile of LCLs is more closely related to that of B cells than WB and, additionally, that LCLs may nevertheless be useful for life-course environmental epigenomics.