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.

Transgenerational epigenetic inheritance: how important is it?

Much attention has been given to the idea of transgenerational epigenetic inheritance, but fundamental questions remain regarding how much takes place and the impact that this might have on organisms. We asked five leading researchers in this area--working on a range of model organisms and in human disease--for their views on these topics. Their responses highlight the mixture of excitement and caution that surrounds transgenerational epigenetic inheritance and the wide gulf between species in terms of our knowledge of the mechanisms that may be involved.

Maternal inheritance of a promoter variant in the imprinted PHLDA2 gene significantly increases birth weight.

Birth weight is an important indicator of both perinatal and adult health, but little is known about the genetic factors contributing to its variability. Intrauterine growth restriction is a leading cause of perinatal morbidity and mortality and is also associated with adult disease. A significant correlation has been reported between lower birth weight and increased expression of the maternal PHLDA2 allele in term placenta (the normal imprinting pattern was maintained). However, a mechanism that explains the transcriptional regulation of PHLDA2 on in utero growth has yet to be described. In this study, we sequenced the PHLDA2 promoter region in 263 fetal DNA samples to identify polymorphic variants. We used a luciferase reporter assay to identify in the PHLDA2 promoter a 15 bp repeat sequence (RS1) variant that significantly reduces PHLDA2-promoter efficiency. RS1 genotyping was then performed in three independent white European normal birth cohorts. Meta-analysis of all three (total n = 9,433) showed that maternal inheritance of RS1 resulted in a significant 93 g increase in birth weight (p = 0.01; 95% confidence interval [CI] = 22-163). Moreover, when the mother was homozygous for RS1, the influence on birth weight was 155 g (p = 0.04; 95% CI = 9-300), which is a similar magnitude to the reduction in birth weight caused by maternal smoking.

Human transgenerational responses to early-life experience: potential impact on development, health and biomedical research.

Mammalian experiments provide clear evidence of male line transgenerational effects on health and development from paternal or ancestral early-life exposures such as diet or stress. The few human observational studies to date suggest (male line) transgenerational effects exist that cannot easily be attributed to cultural and/or genetic inheritance. Here we summarise relevant studies, drawing attention to exposure sensitive periods in early life and sex differences in transmission and offspring outcomes. Thus, variation, or changes, in the parental/ancestral environment may influence phenotypic variation for better or worse in the next generation(s), and so contribute to common, non-communicable disease risk including sex differences. We argue that life-course epidemiology should be reframed to include exposures from previous generations, keeping an open mind as to the mechanisms that transmit this information to offspring. Finally, we discuss animal experiments, including the role of epigenetic inheritance and non-coding RNAs, in terms of what lessons can be learnt for designing and interpreting human studies. This review was developed initially as a position paper by the multidisciplinary Network in Epigenetic Epidemiology to encourage transgenerational research in human cohorts.

Change in paternal grandmothers' early food supply influenced cardiovascular mortality of the female grandchildren.

BACKGROUND: This study investigated whether large fluctuations in food availability during grandparents' early development influenced grandchildren's cardiovascular mortality. We reported earlier that changes in availability of food - from good to poor or from poor to good - during intrauterine development was followed by a double risk of sudden death as an adult, and that mortality rate can be associated with ancestors' childhood availability of food. We have now studied transgenerational responses (TGR) to sharp differences of harvest between two consecutive years' for ancestors of 317 people in Överkalix, Sweden. RESULTS: The confidence intervals were very wide but we found a striking TGR. There was no response in cardiovascular mortality in the grandchild from sharp changes of early exposure, experienced by three of the four grandparents (maternal grandparents and paternal grandfathers). If, however, the paternal grandmother up to puberty lived through a sharp change in food supply from one year to next, her sons' daughters had an excess risk for cardiovascular mortality (HR 2.69, 95% confidence interval 1.05-6.92). Selection or learning and imitation are unlikely explanations. X-linked epigenetic inheritance via spermatozoa seemed to be plausible, with the transmission, limited to being through the father, possibly explained by the sex differences in meiosis. CONCLUSION: The shock of change in food availability seems to give specific transgenerational responses.

The anthropometry of children and adolescents may be influenced by the prenatal smoking habits of their grandmothers: a longitudinal cohort study.

OBJECTIVES: Previously, in the Avon Longitudinal Study of Parents and Children (ALSPAC), we have shown different sex-specific birth anthropometric measurements contingent upon whether or not prenatal smoking was undertaken by paternal grandmother (PGM±), maternal grandmother (MGM±), and the study mother (M±). The findings raised the question as to whether there were long-term associations on the growth of the study children over time. METHODS: Measures of weight, height, body mass index, waist circumference, lean mass, and fat mass of children in the ALSPAC study from 7 to 17 years of age were used. We compared growth in four categories at each age: PGM+M- with PGM-M-; MGM+M- with MGM-M-; PGM+M+ with PGM-M+; MGM+M+ with MGM-M+; and adjusted for housing tenure, maternal education, parity, and paternal smoking at the start of the study pregnancy. RESULTS: We found that if the PGM had, but the study mother had not, smoked in pregnancy, the girls were taller and both genders had greater bone and lean mass. However, if the MGM had smoked prenatally but the mother had not (MGM+M-), the boys became heavier than expected with increasing age-an association that was particularly due to lean rather than fat mass, reflected in increased strength and fitness. When both the maternal grandmother and the mother had smoked (MGM+M+) girls had reduced height, weight, and fat/lean/bone mass when compared with girls born to smoking mothers whose own mothers had not smoked (MGM-M+). CONCLUSIONS: This study indicates that smoking in humans can have sex-specific transgenerational effects.

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.

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.

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.

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.

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.

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.

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.

The burden of disease associated with filaggrin mutations: a population-based, longitudinal birth cohort study.

BACKGROUND: Atopic disease is a major health problem. Mutations in the filaggrin gene (FLG) confer major susceptibility to eczema and related asthma. OBJECTIVE: We sought to determine the natural history and burden of atopic disease conferred by the 2 most common FLG mutations in a large, population-based birth cohort study. METHODS: We analyzed the effect of the most common null alleles (R501X and 2282del4) on several atopic phenotypes in a cohort of approximately 7000 English children born in 1990-1991. RESULTS: FLG null alleles associated strongly with eczema; eczema associated with these mutations presents in early life and is more persistent (hazard ratio for eczema resolution for those with FLG mutations to FLG wild type, 0.67; 95% CI, 0.58-0.77; P = 5 x 10(-8)). FLG mutations conferred a population asthma risk of 1.80 (95% CI, 1.34-2.41; P = .00019); asthma risk was especially high in the context of eczema (odds ratio, 3.16; 95% CI, 2.25-4.43; P = 1.4 x 10(-11)). Strong associations were identified with sensitization to grass, house dust mite, and cat dander and sensitization to multiple allergens (odds ratio, 2.12; 95% CI, 1.03-4.37; P = 5.42 x 10(-27)). CONCLUSION: FLG mutations are strong genetic determinants of eczema, early wheeze, asthma in the context of eczema, and atopic sensitization. They confer risk of a particular trajectory for eczema, with increased duration of disease and greater risk of asthma and multiple allergic sensitizations. FLG alleles help define the risk profile of children with eczema and help define the "eczema plus early wheeze" and "eczema plus asthma" phenotypes.

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.

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.

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.