The aims of this study were to assess whether: (i) a history of either grandmother smoking in pregnancy was associated with any of the four behavioural-trait scores independently predictive of autism in the grandchild or with diagnosed autism in the grandchild; (ii) whether any such relationships varied with the sex of the study child, and (iii) whether the results depended on whether or not the study mother also smoked in pregnancy. We found that two of the four autistic traits in the grandchild (F2) were increased in prevalence if the maternal grandmother (F0) smoked in pregnancy especially if the mother herself (F1) did not herself smoke, but that diagnosed autism was also associated with the maternal grandmother smoking in pregnancy.

For two of the traits (Social Communication and Repetitive Behaviour) we have shown that grand-daughters were much more affected than grandsons, and that the associations were particularly apparent if the mother herself did not smoke in pregnancy. The associations tended to increase in effect size after adjustment for social and biological factors of the grandparents. This is not the first time that a sex-specific association between grandmother’s prenatal smoking and grandchild outcome has been shown. Previous studies from ALSPAC have used child growth as outcomes11,12. These demonstrated strong positive effects on fetal growth when the maternal grandmother smoked in pregnancy – the associations were found with grandsons but not grand daughters. During childhood these boys continued to put on weight, especially lean weight. When the paternal grandmother smoked in pregnancy, there was little effect on fetal growth of the grandchild, but the grandchildren grew taller and larger especially if they were girls. Clearly the relationships between grandmother’s prenatal smoking and grandchild’s growth is complex, especially as the effect sizes tended to increase as the grandchild went through adolescence. For this reason in this study, we extended the study to look at the social communication trait at age 16, but found no difference in effect size compared with the relationships at age 7 (data not shown).

These results are intriguing. There remains the question as to whether they are the result of confounding that has not been taken into account. This seems unlikely since on allowance for the demographic and biological factors, the effect sizes increased rather than decreased. If there was a systematic failure in the number of characteristics taken into account, one would expect similar results for paternal grandmother smoking as for maternal grandmother exposure; one would also expect the same results for boys as for girls. We have shown that, after adjustment, cases of diagnosed autism overall were similarly associated with maternal grandmother’s smoking in pregnancy, and similarly increased risk when the mother (F1) did not smoke, as found with two of four different traits predictive of ASD. Such findings make a causal connection more plausible, although the mechanism warrants further investigation. Unfortunately the numbers of children with diagnosed autism were not sufficient to assess whether there were sex-specific results as found with the two traits.

We only allow for features of the grandparents rather than the parents when considering the traits, since parental attributes may well be on the causal pathway. Indeed a further exercise for this study in the future is to determine whether there are features of the personality and attitudes of the mother that may explain the associations between her exposure in utero and her child’s autistic traits. For diagnosed autism, we are aware that although we used a variety of sources to identify the cases, there are consistent gaps – particularly where the mother had a low level of education, lived in public housing and/or had an external locus of control orientation. We considered it important to take these factors into account because they were likely to have resulted in the lack of a diagnosis even though the criteria were present. Interestingly, only when these factors were taken into account was the association with grandmother’s prenatal smoking revealed.

One statistical criticism may be that we did not allow for missing data. The decision not to do this concerned our doubts as to whether the data were missing at random. We strongly suspect not, and have therefore not employed techniques such as MICE. However it is important to note that the results shown were mostly apparent in both the unadjusted and adjusted results.

There are a number of strengths to this study. (a) The population was defined by expected date of delivery and geographical area of residence; it is therefore not biased by likelihood of having autistic traits. (b) A large number of scales were assessed to determine the likelihood of which were able to best predict ASD; this process involved all children for whom the data were available. (c) The data concerning features of the grandparents were collected during the pregnancy resulting in the grandchild, therefore prior to any suggestion that there was anything amiss with the behaviour or development of the child. (d) All parents were invited to complete all questions in the frequent questionnaires they received, not just those relevant to their child.

However there are a number of limitations: (1) We are relying on the accuracy of the reports by the study parents concerning their parents, and this would have been difficult for those who had difficult childhoods; they were encouraged to contact relatives wherever possible to fill in gaps in their own knowledge (an advantage of having the leisure to complete a questionnaire in their own home). (2) The results are mainly relevant to white grandparents living in Britain, the numbers were too small to subdivide the analysis into different minority ethnic backgrounds. (3) The study was not originally planned to look at autism as, at the time of planning (1988) the prevalence was thought to be so low as to suggest that no more than 10 cases might be included in the study. Consequently sets of trait questions were not designed as measures of autistic traits but rather to identify the child’s performance in regard to a large number of attributes at different ages; regression analyses had identified those related to social communication, coherent speech, repetitive behaviour and sociability as being independently predictive of ASD within the ALSPAC study14. (4) Similarly the questions on abnormal and repetitive behaviour were used post hoc to define an autistic trait, and could be criticised for this.

Some may consider the fact that we have not corrected for the number of tests carried out as a criticism, but it should be remembered that we were testing a specific hypothesis concerning the possibility of prenatal smoke exposure to the F1 generation having an association with autism and autistic traits in generation F2. We had stipulated sex differences and differences between the offspring of women who did and did not smoke. We used nine tests for each of the five outcomes, and demonstrated 15 adjusted associations at P < 0.05, compared with 2 expected if the results occurred by chance.

There are two plausible candidate mechanisms for the observed association of ASD risk with maternal prenatal tobacco exposure; transmission of damage to mitochondrial DNA (mtDNA) or epigenetic inheritance from one generation to the next. Maternal smoking in pregnancy causes mtDNA damage to the newborn15 and the transmission of mtDNA variation particularly heteroplasmic mutations (i.e. coexistence of normal mtDNA and mtDNA with pathogenic mutations in the same cell) is increased in ASD9. Mitochondrial transmission across the generations is exclusively via the mother, so is compatible with our observed associations between maternal prenatal tobacco exposure and adverse scores on Social Communication and Repetitive Behaviour measures in her granddaughters. Typically, a mother transmitting heteroplasmic mtDNA mutations is asymptomatic. A maternal grandmother smoking in the latter part of pregnancy could result in direct tobacco effects on both the developing oocytes of her female fetus, and the recombining chromosomes contained therein that are destined for any grandchildren. There is evidence that mitochondrial transmission acts as a sex-specific selective sieve with mutant mtDNA being better tolerated in females compared to males16. How this relates to the sex-specificity observed in this study is unclear.

Epigenetic regulation of gene expression through DNA methylation, histone modification and non-coding RNAs is recognised as part of an organism’s response to environmental exposures, but how elements of this epigenetic response may be transmitted to the next generation(s) is largely unresolved.

Epigenetic transmission in relation to ASD risk has been explored within high risk families using human sperm that are more accessible than oocytes17. The authors conclude that epigenetic (DNA methylation) differences in paternal sperm may contribute to autism risk in offspring based on the Autism Observational Scale for Infants at 12 months of age. To our knowledge, there are limited published data on human multigenerational studies of epigenetic adjustments in relation to maternal smoking, but there are large DNA methylation studies of children exposed to maternal smoking as a fetus. A recent meta-analysis18 revealed differential methylation at several genes previously associated with ASD. These include the DLGAP2 (SAPAP2) gene that encodes a protein involved in the molecular organization of synapses and in neuronal cell signalling and is known to be linked to ASD19,20; and also the neuropilin-2 (NRP2) gene, polymorphisms of which have been associated with autism21. The association of child DNA methylation with maternal smoking in pregnancy has been studied in ALSPAC with a focus on the persistence of the differential methylation from newborn to age 17 years22 One such persistent altered methylation pattern involved the CNTNAP2 gene that encodes a neuronal transmembrane protein member of the neurexin superfamily involved in neural-glia interactions. A common variant of CNTNAP2 is an established risk factor for autism23,24. Indeed CNTNAP2 variants are associated with some of the traits that predict ASD risk used in this study14, although another study did not find this25. Whilst the above associations lend support for the idea that smoking may induce (directly or indirectly) epigenetic changes in fetal genes relevant to ASD risk, they do little to clarify the nature of the positive association with ASD risk in the subsequent generation. The epigenetic response is likely to be complex. Smoking is associated with DNA damage26 and this in turn may induce the production of non-coding (micro) RNAs that can be transmitted to subsequent generations to target several genes relevant to ASD for silencing27.

We have used exposure of the parent to smoking in utero for our studies of intergenerational effects for two reasons. First this is a habit that is easily remembered; and second, cigarette smoking in pregnancy is known to have an adverse effect on child development, and on DNA methylation – and an effect on the next generation is theoretically possible. The analyses were undertaken to ascertain whether there might be sex-specific associations between autistic traits and parental exposure to smoking in utero, but without prior hypotheses as to which grandmother or child’s sex might be involved; consequently it is particularly important that these associations be confirmed in other studies.