For mediation analyses, we tested 71 CpGs individually as candidate mediators found to be differentially methylated (Bonferroni-adjusted P < 6.94 × 10 −8 ) in crude models and models adjusting for cell-type/DNAm heterogeneity.

The 71 CpGs were annotated to genes enriched (FDR < 0.05) within the notch signaling pathway, pathways in cancer, and the human papillomavirus infection pathway (Web Table 2).

The 71 CpGs associated with smoking were enriched in DNAse I hypersensitivity sites of fetal placental tissues (Web Figure 4).

Genomic annotation and summary results from fully adjusted EWAS analyses of prenatal smoking for the 71 CpGs con- sidered as candidate mediators are reported in Web Table 3.


We found 7 CpGs mediating the association between prena- tal maternal smoking and birth weight (FDR < 0.05; adjusted for 71 comparisons).

Among these 7 CpGs, we observed exposure-mediator interaction for 5 (P < 0.10), as shown in Table 2.

As shown in Table 3, among mediators interacting with prenatal smoking, cg14160212 (located within 151 base pairs of the myelodysplastic syndrome 2 translocation-associated protein (MDS2) gene) had a mediated effect of 280-g lower birth weight (95% CI: −478, −102) for smokers but no mediating effect for nonsmokers.

Similarly, cg22638236 annotated to the pre-B-cell leukemia homeobox 1 protein gene (PBX1) had a mediated effect of 301-g lower birth weight (95% CI: −543, −86) for smokers but no mediated effect for nonsmokers.

DNAm of cg20385913 (within 5,569 base pairs of the cytochrome P450 family 1 subfamily a member 2 (CYP1A2) gene) had a mediated effect of 244-g lower birth weight (95% CI: −457, −49) for smokers and no mediating effect for nonsmokers.

DNAm of cg11280108 (located within 3,609 base pairs of Vpr (HIV-1) binding protein (VPRBP)) had a mediated effect of 185-g lower birth weight (95% CI: −363, −25) for smokers and no mediated effect in nonsmokers.

Last, cg01883283 annotated to the WW domain binding protein 1-like gene (WBP1L) had a mediated effect of 135-g lower birth weight (95% CI: −246, −34) among smokers only.

As noted in Table 2, no exposure-mediator interac- tion was observed for cg23878024, with an average mediated effect of 86-g lower birth weight (95% CI: −144, −34), and Distribution of DNAm levels among the 7 mediating CpGs are shown in raincloud plots (Web Figure 5) and according to smoking status on boxplots (Web Figure 6).

Mediation analysis using birth-weight-for-gestational-age z scores as the outcome, instead of birth weight, yielded consistent results (Web Table 4).

Using 437 paired cord blood samples, we did not observe medi- ated effects at the 7 CpGs (Web Table 5).


For comparability, we examined CpGs previously reported to mediate smoking-birth weight associations by placental DNAm in, to our knowledge, the only EWAS performed to date (16) and observed a consistent average causal mediated effect of 145-g lower birth weight (95% CI: −258, −36) for cg27402634 annotated to the leucine, glutamate, and lysine rich 16), possibly due to single-nucleotide polymorphisms within the probes (cg20340720, cg12294026) that might differ by minor allele frequency (Web Tables 6 and 7).

Another important difference is that we used the MethylationEPIC array covering over 400,000 new CpGs located mostly at regulatory elements compared to the 450K previously used (31).


In sensitivity analyses of the mediated effect estimates, the sequential ignorability assumption might be violated for resid- ual correlations of the mediator and outcome regressions far from the observed estimated mediated effects for all 7 CpGs (Web Figure 7).


Prenatal maternal smoking is an established risk factor for lower birth weight.

Here we report our finding that placental DNAm of genes associated with prenatal maternal smoking mediated the association with reduced birth weight.

We observed interaction with 5 of the 7 CpGs found.

This resulted in a strong mediated effect among smokers and null, attenuated effects for nonsmokers for 5 out of the 7 CpGs.

Our results suggest that DNAm variability in the placenta associated with prenatal mater- nal smoking might play an active role in the well-established association of prenatal smoking and low birth weight.

Our results also highlight the need to consider exposure-mediator interac- tions in epigenetic studies as a relevant biological and statistical model.


We observed several exposure-mediator interactions sug- gesting that prenatal maternal smoking must be present for the Downloaded from https://academic.oup.com/aje/article/188/11/1878/5566169 by Stanford University user on 03 October 2025 mediator, placental DNAm, to affect birth weight.

This is a prime example of a mediated interaction, plausible for biologi- cal mechanisms and models (32).

A mediated-interaction epige- netic model is supported by previous studies that demonstrate epigenetic malleability associated with environmental expo- sures.

For example, we have found that epigenetic variability associated with prenatal environmental exposures at birth might be attenuated as children age (33, 34).

Additionally, in a ran- domized trial it was demonstrated that DNAm variability asso- ciated with prenatal maternal smoking in placenta, cord blood and buccal cells can be restored to "normal" DNAm levels comparable to that of infants born to nonsmoking mothers after vitamin-C supplementation (35).

This reversibility of the medi- ated effect supports the hypothesis that prenatal maternal smok- ing must be present for DNAm to affect birth weight.

This concept of mediated interaction has been proposed to more accurately reflect phenomena observed in models of disease (32).

We propose that a mediated-interaction model is highly relevant to epigenetic studies.


The strongest CpG mediator was annotated to PBX1.

PBX1 encodes a nuclear protein that belongs to the PBX homeobox family of transcriptional factors, widely transcribed throughout the developing murine embryo and essential for skeletal pattern- ing and programming (36).

The PBX1 gene regulates osteoblast differentiation by altering chromatin structures and regulating transcription of bone specific genes (37).

A previous EWAS of cord blood reported greater DNAm of a genomic region in PBX1 to be strongly associated with higher birth weight for gestational age (38).

In adults, higher urinary nicotine equiva- lents among smokers were associated with greater leukocyte DNAm at CpGs near PBX1 (39).

In a large study of adults, PBX1 DNAm of leukocytes associated with current body mass index across multiple cohorts, and this association was repli- cated for DNAm of adipose tissue (40).

Whether or not PBX1 DNAm of placenta at birth is relevant for future child adiposity remains to be tested.


Prenatal smoking was also associated with 6.3% lower DNAm at cg01883283 annotated to WBP1L and mediated asso- ciations with birth weight.

Placental DNAm of this gene has been linked to prenatal smoking at a nearby CpG within 145 base pairs of our site (8.7% lower DNAm at cg20340720) (16).

However, in this previous study, no evidence of a mediated effect between prenatal smoking and birth weight was found for cg20340720.

Another CpG mediator, cg24005876, annotated to the cell division protein kinase 6 (CDK6) gene, is member of the serine/threonine protein kinases family and a critical regulator of cell cycle.

The CDK6 gene has been shown to play an important role in the development of polyploidy as well as stromal decid- ualization (41).

In a study of human term placentas, CDK6 mRNA was modestly correlated with infant birth weight (r = 0.33) (42).

This gene also covers a susceptibility loci for lung cancer shown to interact with smoking status/history (43).


CYP1A2 is involved in the metabolism of pharmacologi- cal compounds as well as polycyclic aromatic hydrocarbons (PAHs) present in cigarette smoke (44).

The CYP1A2 enzyme is induced by tobacco smoke, shown to be primarily mediated by aryl hydrocarbon receptor ligands like polycyclic aromatic hydrocarbons (45).

Placental CYP1A2 expression has been de- tected in first trimester but not full-term placentas (46).

From candidate gene studies, lower placental DNAm of the CYP1A1 gene, another CYP family member, has been consistently asso- ciated with prenatal smoking (47-49).

Additionally, prenatal maternal smoking has been consistently reported to be associ- ated with differential DNAm near CYP1A1 in cord blood analyses, as well as in an EWAS of placenta (9, 16).


Less is known about DNAm of cg11280108 within 3,611 base pairs of the Homo sapiens VPRBP gene but this region has been shown to be involved in cell proliferation, chromatin remo- deling, and oocyte survival and programming (50-52).

Much less is known about DNAm or placental expression of cg14160212 (myelodysplastic syndrome 2 translocation-associated pro- tein (MDS2)) or cg23878024 (CD28), and these CpGs did not directly annotate to genes.

The underlying genetic archi- tecture could also influence observed results.

However, only one of our mediators (cg01883283; WBP1L) was located within 500 kb of a birth-weight single-nucleotide polymorphism (rs74233809) (53).


A previous study performed an EWAS of prenatal mater- nal smoking and placental DNA, investigating the mediated effect of DNAm (16).

In this study, cg27402634 (LEKR1) ex- plained up to 36% of the association between prenatal smoking and lower birth weight.

In our cohort, cg27402634 had the strongest association with prenatal smoking but did not reach an FDR < 0.05 for the average mediated effect.

We did observe a similar mediated effect of 145-g lower birth weight post hoc, but contrary to the previous study, an exposure-mediator inter- action was present at this site.

Although we did not observe mediation for the other 2 CpGs reported by Morales et al. ( 16) (cg25585967 and cg12294026) within the trio rho guanine nucleotide exchange factor (TRIO) gene body, they were con- sistently associated with greater placental DNAm for smokers with similar magnitudes.


Our results highlight that prenatal maternal smoking signatures of placenta DNAm are distinct of cord blood signatures.

For example, using paired cord blood samples from our study we found no evidence of mediation at the 7 discovered CpGs from placenta.

We compared the 71 CpGs associated with maternal smoking in placenta to 6,073 CpGs (8) previously reported from meta-analyses of cord blood and found that only 1 CpG (cg21992501; tetratricopeptide repeat domain 27 (TTC27)) positively associated with smoking in our placenta samples and in the meta-analyses of cord blood.


Our study has several important limitations.

In DNAm studies with exposure misclassification, it has been demonstrated that DNAm of CpGs could serve as better biomarkers of the exposure than self-report, thereby increasing the type I error rate, underesti- mating the direct effect, and overestimating mediated effects (54).

We also did not collect objective exposure biomarkers, and it is possible that our estimated mediated effects are inflated.

However, self-reported smoking during pregnancy had high sen- sitivity (97.6%) and specificity (100%) compared with plasma cotinine in a comparable Canadian population (55) and other large cohorts (56).

Another important limitation is that we did not ascertain sustained smoking throughout pregnancy-women could have stopped smoking after the first research visit-or ex- posure to secondhand smoke.


Future studies should consider the impact of periconceptional paternal smoking and its impact on germ cell DNAm and on the infant's epigenome.

Timing of ex- posure, mediator, and outcome ascertainment is an important limitation of our study-women could have continued to Downloaded from https://academic.oup.com/aje/article/188/11/1878/5566169 by Stanford University user on 03 October 2025 smoke throughout pregnancy and placental DNAm levels could have changed until birth, when birth weight was mea- sured.

However, in the general Canadian population, approxi- mately 53% of women quit smoking by the third trimester of pregnancy (57), so it is likely that most women in our study quit by the third trimester.

Additionally, placental DNAm profiles are largely established in early gestation.

Our assumptions are that smoking in early pregnancy influences DNAm of placental genes that are established in early gestation, thereby influencing fetal growth.

Nevertheless, it is possible that fetal growth influences placenta DNAm.

Therefore, our results must be interpreted in light of the timing of exposure and DNAm measurements.


An important strength of our approach is the implementation of the counterfactual framework in mediation analysis to esti- mate effects in the presence of exposure-mediator interactions as a relevant biological model for epigenetic epidemiology.

Nevertheless, statistical mediation might not accurately reflect underlying causal biological processes such as transcriptional memory or cell lineage commitment.

Therefore, our results do not necessarily reflect a causal biological mechanism.

Future studies could also develop epigenetic biosensors of prenatal smoking from placenta DNAm signatures that might serve as biomarkers of the prenatal environment.


In conclusion, in this cohort, we observed that placental DNAm variability at specific loci associated with prenatal mater- nal smoking might mediate the association with lower birth weight within biologically relevant genes.

Placental DNAm mediators annotated to the PBX1 gene, involved in skeletal pat- terning and programming; CYP1A2 gene, whose activity is induced by polycyclic aromatic hydrocarbons found in cigarette smoke; CDK6, whose expression in placenta has been associ- ated with birth weight and stromal decidualization; and the WBP1L gene, previously shown to be differentially methylated in the placenta of smokers.

Finally, we report evidence of signifi- cant exposure × mediator interactions and propose this to be highly relevant model for epigenetic epidemiology.


A substantial body of literature has shown robust associations between prenatal smoking exposure and DNA methylation levels.

The pattern of DNA methylation can be used as a molecular signature of past prenatal smoking exposure and might also provide mechanistic insights into associations between prenatal smoking exposure and adverse health outcomes.

In this issue of the Journal, Cardenas et al. [ 6] evaluated whether DNA methylation mediates the association between prenatal smoking and low birth weight in a tissue that is mechanistically relevant to birth weight—the placenta—using formal mediation analyses.

They found that methylation levels, at 5 loci, mediated smoking exposure effects on birth weight but only among children whose mothers smoked during pregnancy.

Given the use of formal mediation methods and measurement in a mechanistically relevant tissue, this work has the potential to inform novel directions for intervention.

Replication of these findings in larger and more racially and ethnically diverse samples, repeated measures to better tease apart the timing of DNA methylation changes with respect to exposure and birth weight, and continued use of intervention-focused mediation methods are needed before the impact of these findings will be fully realized.


Since the publication of Joubert et al. 's [1] seminal work showing a replicable epigenetic signature of prenatal smoking exposure in the cord blood of newborns, epidemiologists and pediatricians, among many others, have been excited about the potential implications.

Joubert et al. performed an epigenome-wide association study (EWAS) that identified DNA methylation at 26 specific loci across the genome that showed statistically significant differences in cord blood methylation among infants from Norway between those whose mothers smoked tobacco during pregnancy and those whose mothers did not smoke.

The methylation effect estimates for these same 26 loci were remarkably similar in cord blood samples from infants in North Carolina, showing a measurable biological impact of prenatal environmental experiences that can be detected after birth.

This was reinforced by later work showing similar findings in peripheral blood collected during early childhood, adolescence, and adulthood [2-5].

These highly robust association findings between prenatal smoking exposure and DNA methylation at birth or later in life are exciting for 2 reasons: 1) they suggest that environmental biomarkers of prenatal exposures can be measured from samples obtained much later than the prenatal window, thus expanding opportunities to examine prenatal origins of health using cohorts and other samples for whom exposure information was not obtained during that window; and 2) they offer the possibility that DNA methylation changes might mediate prenatal exposure effects on child health outcomes.

A methylation-mediated mechanism could further inform prevention strategies beyond existing guidelines to cease smoking during pregnancy or could inform strategies to ameliorate harmful effects in the child after exposure.


However, several key issues must be addressed when considering mechanistic implications of methylation, including tissue specificity, timing, and design and analytical tools applied to specific mechanistic questions.

A first consideration is that DNA methylation patterns are tissue- and cell-type specific, such that information from blood samples might not be relevant to mechanisms that involve other tissue types.

This is part of the purpose of epigenetic machinery: to define the pattern of genes that should be “on” or “off” in particular cells and organs.

Therefore, blood-based DNA methylation measurement might not be a direct indicator of exposure-disease mechanisms if blood is not a primarily involved tissue.

Epigenetic measurement in other tissue types would be necessary.

Second, epigenetic marks are dynamic over time.

This provides a mechanism for cells to respond to their environment and to control key developmental functions (e.g., so that a fetal brain grows rapidly but doesn’t continue to grow in adulthood).

Thus, timing of the epigenetic measurement is critical to a mechanistic interpretation.

Given these caveats, the robust previous findings of association between a prenatal smoking exposure and infant or childhood DNA methylation in blood might not necessarily inform the mechanism of risk for outcomes where blood is not the primary affected tissue or when methylation state at birth—or at a particular postnatal age—might not be relevant to the mechanistic timing of epigenetic influence.

Mechanistic insights require examination of the epigenetic patterns in specific tissues and specific developmental windows.


Beyond these considerations, there has to date also been a paucity of appropriate design and analytical approaches that specifically evaluate DNA methylation as a mediator of exposure-outcome associations.

Previous studies have shown associations between prenatal smoking and DNA methylation, and separately between DNA methylation and child health outcomes, including preterm birth and birth weight, yet these have rarely been examined together via formal mediation analyses and with appropriate prospective data collection.


In this issue of the Journal, Cardenas et al. [ 6] address these limitations by estimating causal mediation effects of DNA methylation in the relationship between prenatal smoking exposure and low birth weight in the child.

Instead of cord blood, they measured DNA methylation in placenta, a developmental tissue highly relevant to fetal growth.

They tested 720,077 loci in the genome and found statistically significant methylation differences between smokers and nonsmokers at 71 of those loci.

They further tested for epigenetic mediation using the mediated interaction decomposition methods of VanderWeele [7] and reported mediated interaction at 5 specific locations in the genome (cytosine-phosphate-guanine (CpG) sites), where the mediated association of maternal smoking through methylation to birthweight was detectible only in the presence of maternal smoking.

These results suggest that methylation levels at specific CpG sites, in a tissue that is directly relevant to birth weight, might mediate the effects of prenatal smoking on birth weight.

If true, this would suggest methylation as a potential target of intervention to prevent low birth weight among mothers who smoke in pregnancy.


It is encouraging to see an application of formal mediation analysis in this context, particularly for a growth-relevant tissue type, and measurement of exposure, methylation, and outcome in a plausible timing sequence to infer directional mediated effects.

The potential importance of this work would be the implication that methylation could serve as a biologic target for future prevention/intervention efforts to prevent low birth weight, particularly among mothers who smoke.

Because DNA methylation is modifiable, there is some promise to this line of thinking.

However, much is left to do before the promise of such a finding can be realized.

As with all moderately sized genome-scale discovery approaches, larger samples and greater precision of effect sizes is needed.

Assuming that epigenome-wide association study results trend similarly to analogous genome-wide association study (GWAS) pursuits, much larger samples will be needed to define reliable effect sizes.

Further, there are likely many placental CpG sites that truly associate with prenatal smoking and/or with low birth weight that could not be detected in this current sample.

Consistent, yet small, risk effects in GWAS have tended not to emerge until tens to hundreds of thousands of samples were included in pooled or meta-analyses [8].


Like genetic studies, DNA methylation studies are also susceptible to confounding by ancestry as well as to heterogeneity of effects by ancestry and environment.

The sample in Cardenas et al. is restricted to one ancestry and cultural/environmental context, so it might not be generalizable to different genetic backgrounds or prenatal environmental contexts.

Indeed, few of the previously reported placenta DNA methylation CpG sites associated with maternal smoking [9] showed similar effect sizes in this current sample, potentially due to differences in context.

This highlights the need for larger and more diverse samples in future discovery and replication studies, while also protecting against confounding by ancestry among other sources of confounding.

Race- and ethnicity-specific findings will be important to inform population-relevant intervention strategies and to ensure equity in this type of research.


There are a few other points to consider that might be fruitful in future directions.

First, there is evidence that methylation might act in coordinated ways, such that a single CpG site is less relevant than a group of nearby, or even translocated, CpGs.

This would imply that, rather than considering each CpG in a separate set of association and mediation analyses, machine-learning or spatially informed sets of CpG methylation might be the best unit of inquiry.


Second, the analytical approach used by Cardenas et al. [ 6] provides strong statistical evidence for mediation effects but does not overcome concerns regarding the timing of the exposure, DNA methylation, and birth weight measures.

Methylation in placenta is essentially measured at the same time as the outcome, although it presumably reflects recent methylation states during pregnancy.

The same can be argued for the timing overlap of exposure and placental methylation.

The lack of longitudinal measures for each these factors over the course of pregnancy makes true causal inference hard.

Emerging methods using extracellular vesicles that originate in the placenta might allow this for other forms of epigenetic marks, such as microRNAs, which can be measured in real time in maternal circulating blood during pregnancy [10-12].


Finally, the specific approach to mediated interaction estimation is worth discussion.

In the DNA methylation literature, several groups have applied the “causal inference test” approach from the gene expression literature [13, 14], where the goal is to rule out associations between DNA methylation and disease outcomes that are likely consequences of the disease rather than causes—particularly when using case-control designs.

These were developed for mediation that assumes that methylation mediates genetic associations with outcome, similar to Mendelian randomization.


The prenatal smoking exposure of interest in Cardenas et al. [ 6] cannot rely on such an assumption of directionality or absence of confounding.

This ambiguity and potential for confounding exists between the exposure-mediator relationship and the mediator-outcome relationship in this particular setting.

Further, the main question of the paper is not just whether DNA methylation associations are causal or consequential of low birth weight but rather whether methylation specifically mediates the known association between maternal smoking and low birthweight.

This is exactly where more formal epidemiologic mediation methods, such as those used by Cardenas et al., can be most useful.


However, even in this context, it is important to be very clear about the reason for estimating mediation effects.

Presumably, it is to identify the most efficacious, feasible, and practical target(s) for intervention.

Application of 4-way decomposition and other traditional parametric model–based mediation methods, which aim to determine how much of the total exposure-outcome effect is explained by the mediator, might not be the most informative approach if the goal is to assess potentially modifiable interventions [15].

Nguyen et al. [ 15] have recently described the causal mediation frameworks through this lens, and further work addressing this specific question might benefit from their framing.


In summary, it is clear that DNA methylation measured at birth or after, in blood or other tissues, is associated with maternal smoking during pregnancy.

This has clear utility as a biomarker tool that might enable epidemiologic inquiry about prenatal exposure risks for later disease even when the prenatal window cannot be captured prospectively.

However, the arguably more important implication, that DNA methylation actually biologically mediates the risk effects of prenatal exposure, has yet to be conclusively determined.

Cardenas et al. provide evidence for the potential mediating effects of placental DNA methylation in the risk association between prenatal smoking and low birth weight.

This is promising given the relevance of placenta for fetal growth and the potential modifiability of methylation or associated functional gene regulation.

Yet our field must be vigilant about getting this right by continuing to consider the implications and limitations of evidence to date, given limited sample sizes, knowledge of how DNA methylation works in concert at multiple sites of the “methylome” and with other epigenetic mechanisms, and what can be learned from specific cells and at specific developmental windows, and we must continue to apply rigorous design and analytical approaches with a framework of interventional applications in mind.


We thank Ladd-Acosta and Fallin for their thoughtful commentary [1] regarding our work on prenatal maternal smoking and placenta DNA methylation mediators [2]. They raise several critical points regarding biomarkers and mediators in epigenetic epidemiology.

As noted in their commentary, a promising feature of epigenetic research is the malleability of some epigenetic markers as potential biological targets that might offset adverse health outcomes.

This was in part our motivation in testing epigenetic mediators, in addition to expanding our molecular understanding of the genomic fingerprint of prenatal smoking on fetal growth.


While we agree that much work is needed to capitalize on this promise as a public health intervention, there is encouraging preliminary evidence that supports nutritional interventions that offset epigenetic toxicity associated with environmental exposures.

For example, a randomized crossover trial of subjects exposed to fine particulate matter demonstrated that B-vitamin supplementation nearly prevented DNA methylation alterations compared with placebo [3].

For prenatal smoking, a randomized controlled trial of vitamin-C supplementation showed that the intervention group was able to restore DNA methylation levels of candidate genes associated with prenatal maternal smoking [4].


As suggested, studies in ethnically diverse populations with larger sample sizes and increased statistical power are needed to detect moderate to small associations, likely missed in our study.

Our study was limited in size, within a relatively homogeneous population.

Indeed, for these efforts, we encourage investigators and cohorts with available data to participate in the Pregnancy and Childhood Epigenetics (PACE) consortium, a global consortium of researchers pooling data in meta-analyses of DNA methylation studies across several cohorts [5].


We concur that refining the timing of measurements for both the molecular marker and prenatal maternal smoking are needed to obtain better estimates and avoid cross-sectional comparisons.

In addition, the integration of multiple molecular markers (e.g., genetics, epigenetics, transcriptomics, and proteomics) is needed to fully understand the interplay and downstream consequences of the DNA methylation associations found for prenatal smoking across tissues.


A major challenge highlighted by the authors is the tissue specificity of epigenetic marks and the need to use disease- and exposure-relevant tissues or cells [1].

While blood, cord blood, and now placental DNA are widely collected and being used in epigenetic studies, the tissue-specificity challenge presents an opportunity to study target epigenetic marks relevant to the question.

In addition to asking causal questions, tissue-specific DNA methylation signatures might persist years after an exposure, enabling the discovery of biosensors of environmental exposures [6].


Last, the use of parametric and other statistical mediation analyses has been a topic of debate recently [7].

As pointed out by the authors, it is important to identify the question before performing mediation analyses [1].

Our intent was to answer an explanatory question as well as identifying methylation sites that were significant mediators of the association between maternal smoking and birth weight.

As others have pointed out, a DNA methylation mediator could be a proxy for the causal change and not necessarily a driver of the association.

For example, DNA methylation changes might reflect altered transcription binding [8] or serve as surrogate markers of cell type or cell subtype expansion [9].

We concur that multiple lines of evidence are still needed to provide conclusive evidence and design public health interventions to target epigenetic biomarkers.

Replication across studies, methods, and robustness of results are required before interventions can be recommended.


Critical objections are a welcome means of removing ambiguities or clarifying implicit assumptions.

For this reason, I am very grateful that Luis Cordeiro-Rodrigues has written a reply to my paper "Animal Suffering, God and Lessons from the Book of Job" (Gasser 2021).

This gives me the opportunity to rethink this topic again and try to present it in a more precise manner.

First, I present a brief sketch of my account.

Then I address two points of criticism that seem to be based on misunderstandings.


Central to the problem of evil is the presupposition that God is a morally perfect being.

The core of the discussion works with the following principles:


(a) A morally perfect being eliminates evil as far as it is possible.


(b) Within the realm of logical possibilities, an omnipotent being has no limits of agency.


With these two principles at hand, one can argue that we should expect that a morally perfect and omnipotent being will eliminate all evils unless it is impossible to do so. Since there are many evils in the world that we are certain it is logically possible to avoid or eliminate, we have very strong evidence that such a being does not exist.


Advocates of theism have mainly responded to this challenge by pointing out that there might exist possible higher goods (e.g., freedom of will, character building, union with God, etc.), which can only be achieved by allowing evils in the world.

As a consequence, God is morally justified in permitting these evils as they are necessary and indispensable instruments for bringing about higher goods.

Once this instrumental relation between evils and higher goods is not exclusively reserved for human beings but applied to creation in general, then also the suffering of animals can be explained in a similar fashion.

Trent Dougherty, for example, develops an expanded soul-making theodicy for animals: Accord- ingly, God, by being in a caring and loving relationship with all of creation, will enfold the suffering of any creature in a greater good that organically defeats this evil.

The most likely way to do this is through some future process of soul-making which requires animal resurrection and deification in the eschaton (Dougherty 2014, pp. 145-46).


In a recent paper (Gasser 2021), I argue that this understanding of God is couched in overly personalistic terms.

God is imagined similar to a human person and judged accordingly with the moral standards appropriate for human agents.

Without doubt, there Religions 2022, 13, 264.

https://doi.org/10.3390/rel13030264 https://www.mdpi.com/journal/religions is this tradition of a personal conception of God; however, it should not be ignored that there is also another powerful tradition, namely that of so-called classical theism, in which personal attributes of God are of secondary importance.

This is so because God as creator is not a part of the created world, and therefore God cannot belong to the moral community as humans do.

A main problem of the problem of evil is the anthropomorphic understanding that the human-human relationship is the template for all kinds of relationships, including the divine-human one.


Once this view is rejected, the problem of evil changes as the central moral assumptions regarding God ought to be revised.

We cannot apply our moral categories to God as these categories belong to the created but not to the transcendent order of being.

The idea I am proposing is that God is interested in its creation, but the essential values that shape the divine view are to be explicated less in terms of moral categories than in terms of non-moral goods such as complexity, fecundity, creativity, adaptability, or the will to live.

To put it more pointedly, humans might be less central to God's overall creational purposes than personalistic interpretations of a theistic God generally presuppose.

Such a view may seem odd at first glance, but-according to my argumentation, which draws also upon exegetical interpretations such as those of Carol A. Newsom (Newsom 2003) and Steven Chase (Chase 2013)-the divine speeches in the Book of Job can be understood in precisely this way: God teaches Job to expand his focus by opening up the narrow circle of human-oriented moral questions toward the astonishing and mysterious sublimity of natural and cosmic processes where moral categories are non-applicable.


Both perspectives are separated by the moral sphere as it distinguishes the human and the non-human realm: Responsibility, justice, empathy, or orientation toward moral values are essential coordinates of the human world.

These features constitute the human life-form in the first place.

When it comes to the world of other living beings or the non- living dimensions of creation, however, these features have no essential role to play.

Most scientists consider animals to be a-moral because they lack the cognitive capacities required for (full) moral agency (see, for instance, Tomasello 2019).

Accordingly, we do not blame a predator for killing a pray because hunting and killing animals is a central feature of a predator's life-form, but moral understanding is not.


In the light of this understanding, the divine speeches in the Book of Job highlight both perspectives and the tension that comes with them.

Newsom writes: "What Job has just heard in the divine speeches, however, is a devastating undermining of his understanding of the unproblematic moral continuity between himself, the world, and God. It is a profound loss of unity, a recognition of the deeply fractured nature of reality" (Newsom 2003, p. 253).


There is not one perspective from which the world is grasped as unified and well- ordered but a holistic and an individual perspective, each of which is shaped by different normative schemes.

To a structurally similar conclusion comes philosopher Wes Morriston: "The Hassidic teacher, Rabbi Bunam, said that 'A man should carry two stones in his pocket.

On one should be inscribed, "I am just dust and ashes".

On the other, "For my sake was the world created". And he should use each stone as he needs it.' The experience of the whirlwind has taught Job to use the first stone. But what we need, and what the book of Job tries, with only partial success, to teach us, is how to use them both together" (Morriston 1996, p. 356).


Both Newsom and Morriston point out that the tension in such an understanding of the God-world relationship cannot be brought to a higher synthesis; rather, it is our lot to endure it.

In his encounter with the divine, Job realizes this essential feature of the world and our tragic place in it.

If this assessment of the human condition is correct, then it also helps to understand the criticisms that have been raised in Cordeiro-Rodrigues's reply.


I briefly address two points of criticism that seem to me particularly worthy of con- sideration: The first criticism says that the God I am proposing is immoral because God apparently accepts gratuitous (animal) suffering.

However, "God as creator is also the father of all existing beings and therefore has stronger duties towards them" (Cordeiro- Rodrigues 2022, p. 3) than parents have toward their children.

The second criticism says that if God as a transcendent being is not subject to moral rules, then either no objective moral standards exist or they are inscrutable for us.

In either way, our moral order would be permanently undermined because divine morality does not correspond to ours, and, as a consequence, humans would easily get into situations where they would make the wrong decision and thus be responsible for moral evils (Cordeiro-Rodrigues 2022, p. 6).


The first criticism presupposes that God ought to be thought of as strictly personal and therefore as a moral subject observing those moral rules that are also applicable to us.

Cordeiro-Rodrigues refers to Desmond Tutu's work for making the point that God cares about right and wrong and, thus, about a moral universe.

In analogy to the obvious moral principle that letting someone suffer is only permissible in the light of a higher good attained through it, God has to respect this principle as well.


This interpretation is undoubtedly widespread in current accounts of theodicy.

Take, for instance, Eleonore Stump's remarkable reading of the book of Job in her seminal study Wandering in Darkness (Stump 2010).

She interprets the divine speeches as expression of God's providential care which is directed toward intimate second-personal closeness to God.

Stump concludes: "Focusing on the second-person account of the divine speeches and the second-person experience of God that Job has while God is speaking to him shows us that there is for Job a second-personal explanation of his suffering [ . . . ]" ( Stump 2010, p. 225).


One has to notice, however, that this line of interpretation faces serious challenges.

In her specific account, Stump has to radically reinterpret the many passages of the divine speeches where a wild and untamable nature is presented, which renews itself through the cycle of eating and being eaten.

Here the experience of a second-personal and caring relationship of God to creation finds hardly any point of connection.


In more general terms, the question arises to what extent higher-good theodicies can convincingly explain that all the suffering in the world is a necessary and indispensable means for achieving these goods.

Stump, for instance, explicitly wants to limit her account to adult and mentally functional humans as she does not know whether it is applicable to the nature of suffering of animals, infants, or cognitively disabled individuals and the possibility of their personal growth of the relationship with God (Stump 2010, p. 4; similarly McCord-Adams 1999, p. 28).

A positive evaluation might suggest that there are possibilities of adapting the proposed solution.

However, a negative evaluation will interpret this delimitation as a kind of capitulation in front of particularly challenging instances of suffering.


I think we have a strong intuition to assume that if suffering is necessary at all, then less terrible instances of it would do it as well.

Daniel Howard-Snyder expresses this idea as follows: "Suppose God had simply prevented us from ever having genocidal thoughts. Would we then have been unable to perceive the hideousness of living unto ourselves? Would we have lacked the requisite incentive to turn to God? [ . . . ] We each need to answer this question for ourselves, but for my own part, on careful reflection I can't see how [ . . . ] God would be justified in permitting so much horrific evil and suffering rather than a lot less" (Howard-Snyder 2008, p. 340).


In other words, the effort to understand God as a moral being can lead to the idea that this conception must be abandoned, and God ought to be disqualified as immoral.

To show that God can have good reasons for allowing terrible suffering does not yet imply that God also has good reasons for permitting any kind of suffering, no matter how horrific.

It is precisely the existence of such evils that makes it so difficult to maintain a comprehensive moral order in which God functions as the source and universal guarantor of it.

This difficulty is also evident in animal theodicies when additional assumptions about possible future developmental scenarios of animals in the eschaton are proposed for avoiding the impression of gratuitous animal suffering.

Thus, the focus on a Tutuist framework for tackling the problem of evil constraints the conceptual resources of the debate considerably because moral standards for human-human relationships are elevated to the general standard for any relationship, including the one between God and humans.

The assumption of such a comprehensive moral order of reality entails a considerable argumentative burden, which is often seen as doomed to failure.

It seems to me that Cordeiro-Rodrigues did not adequately grasp this point or he considers it to be as too easily solvable.


The image of God outlined in the Book of Job (and what I have sympathies for) places God beyond such a moral order.

This does not mean that God has no interest in human beings and their well-being; rather, it says that God wants to liberate human beings from the misleading assumption that the entire cosmos is governed according to a moral order, which guarantees an adequate compensation for all instances of suffering.

Such a moral order, to put it bluntly, is an important and worthwhile ideal of human life but, apparently, not of divine creation.

With such an understanding of the God-world relationship, the unity of a moral order encompassing God and creation is no longer available.

By letting the idea of morality as a leading category for our overall understanding of reality go, the human view is able to open up into a profoundly tragic dimension of our existence.

Thus, Newsom draws the following conclusion of her interpretation of the Book of Job: "Like the ostrich, Job, too, brings his children into a dangerous world, where they may well be crushed and trampled.

Unlike the ostrich, however, which forgets the dangers to its offspring, it is unlikely that Job can be said to forget their vulnerability" (Newsom 2003, p. 258).


This brings me to the second criticism, which says that if "there is an inscrutable moral code for humans, then [ . . . ] this would mean that humans can never be held accountable for moral wrongs" (Cordeiro-Rodrigues 2022, p. 3).

Even if there were a divine moral code that is beyond human comprehension, this would not imply that we are bereft of any insight into valid moral principles.

Since one can only use what they have got, in such a scenario, we would have to adhere to the moral principles accessible to us thanks to our reason.

Take the moral insight that suffering may only be imposed on others if this is the only way to achieve a higher good.

This insight is not simply given to us, but we can justify it and argue that it is reasonable.

If, therefore, a hidden divine moral code completely different from our moral principles were to exist, then this matter of fact would not further affect us, since any access to this divine order is denied to us.


Note that the view I am proposing is not making any claims in this direction.

Rather, the idea is that there are non-moral values such as continuous creativity, remarkable adaptability, prolific life-circles, amazing bio-diversity, etc., whose axiological weight we are able to grasp.

If God were to appreciate these values, then we were able to grasp at least certain aspects of this holistic divine perspective on the world.


A final point: Is this concept of God proposed still a Christian God? This question shapes the criticisms put forward. I believe this concept of God belongs to the rich bib- lical tradition which emphasizes that the world as we know it is dangerous.

After all, a single misstep in paradise brought humanity into the world as we know it.

Against this background, the crucial question is: Can it ever be reasonable for participants in terrible suf- ferings like Job to stop seeing this suffering as an obstacle to trusting that God is interested in our well-being and in a relationship with us?


Here is a proposed answer: Those involved in suffering will stop blaming God for it when they accept their lives as they are-even with all the suffering.

This, according to the interpretation I follow, is the lesson that the story of Job wants to teach us.

We come to accept our lives not when we give up all hope but when we reach a point where participation in terrible situations of suffering no longer threatens the integrity of an individual's personality.

Acceptance does not mean letting go of the conviction that suffering was prima facie ruinous to one's life, but it does mean letting go of the demand that things should have gone differently.

Acceptance does not mean that the painful, wrong, and prima facie ruinous events count for nothing or that we should no longer care about them.

Rather, these events are no longer capable of destroying our integrity as a human person because we have reached a state where we no longer have to struggle with what has happened and where we no longer try to find a hidden meaning or divine plan where there is none.

The aim is to come to a stable understanding of our place in the cosmos that is able to integrate what has happened into a meaningful context.


From a Christian perspective, one can add that the firm trust that God is our secure fundament finds personal involvement in suffering terrifying (think of Jesus in Gethsemane) but achievable as it involves the hope that the creator of this world can at the same time be the re-creator of a better world.

Such a view of hope for the next life does neither explain suffering away nor dispose of a grand theory of suffering, but it makes it, at best, bearable.


Georg Gasser has recently attempted a new explanation to the problem of animal suffering, i.e., how can a morally perfect, omniscient, and omnipotent God allow the gratuitous suffering of animals? His argument can be interpreted in two ways: (i) creation is amoral and therefore there is no problem of animal suffering; (ii) God’s morality is beyond us and not responsive to humans.

In both cases, the problem of animal suffering is, according to Gasser, explained.

Grounded on the thought of Desmond Tutu, I contend, however, that both (i) and (ii) imply that God would be immoral, which is an unacceptable implication for Christians.

Therefore, Gasser’s explanation fails to solve the problem of suffering.

Further, I uphold that if God exists He is necessarily a moral agent and if one wishes to give up such property, then also needs to give up His omnipotence.

On top of this, I challenge the idea that there is a naturalistic fallacy in holding a Tutuist conception of God.


In a recent article in Religions, Georg Gasser [1] attempts a new explanation of the problem of animal suffering: how can a morally perfect, omniscient, and omnipotent God allow the gratuitous suffering of animals? This is a challenge to God’s existence because a God with such characteristics would presumably not allow such gratuitous suffering.

Gasser’s answer is slightly ambiguous, and it can have two possible interpretations: (i) animal suffering does not challenge the existence of God because creation is amoral and He does not rule the universe according to moral laws; alternatively, Gasser may be arguing that (ii) the moral laws of God are non-humancentric and thereby not only are not responsive to humans but also beyond human understanding.

Hence, animal suffering does have a moral justification but just not one that humans expect or can understand.

I believe that Gasser argues for (i), but because there are elements in his text that suggest (ii), I will address both in this comment to his article.

Inspired by the thought of Desmond Tutu, I contend that both (i) and (ii) would imply that God is immoral rather than amoral.

More precisely, God’s failure to stop evil He is able to stop at a low cost is immoral because it violates social harmony and, thereby, neglects the moral status of some of His creation.

Additionally, I contend that Gasser’s argument removes the possibility of attributing moral blame or praise to human action, which is not only a questionable moral implication but also an idea at odds with Christian values.

As this is an unacceptable implication for Christians, I argue that Gasser’s theory fails to solve the problem of animal suffering.

Further, I contend that these criticisms I raise do not fall in the naturalistic fallacy.

As a starting point, I treat the problem of evil as fundamentally a moral question [2]; further, I share the intuition of philosophers like Eleonor Stump and Marilyn McCord Adams, that to be sound, a theodicy needs to be grounded in Christian values [3][4].

Note also that my goal is not to challenge the existence of God as I do not carry out an extensive analysis of different theodicies, but simply to challenge Gasser’s argument.


The first possible interpretation of Gasser’s argument is that animal suffering can be explained if God is conceived of in less personalistic terms.

Gasser suggests a God inspired by Classical Theism, where God is a non-personal being.

Thus, God is primarily the creator and sustainer of everything.

So, for Gasser, God is not a guarantor of moral rules and there are no moral principles in creation.

In Gasser’s words: ‘God directly [is] a sublime power in nature who does not rule the world in accordance with a moral order.

’ [ 1].

Thus, if creation is amoral because God is not concerned about morality, then animal suffering is to be understood as a mere fact of creation without needing further moral explanations for why it occurs.

Gasser thereby concludes that ‘the idea is that creation is not ordered according to any principles of justice and therefore it makes no sense to demand them’ [1].

Inspired by the thought of Tutu, I wish to show that Gasser’s argument would lead to an implication that Gasser and most Christians are not willing to accept; that is, if it is true that evil occurs gratuitously and God is able to stop it, but because He does nothing, God is not simply amoral as Gasser suggests.

Instead, if this were the case, God would be immoral.


To understand this, it is important to start by pointing out that for Tutu, we live in a moral universe with a personalistic God.

Indeed, he states that ‘This is a moral universe ( . . . ) God is a God who cares about right and wrong.

God cares about justice and injustice’ [5].

During Apartheid, Tutu wrote to P. W. Botha (who was President of South Africa at the time) telling him that the anti-Apartheid activists have already won because they were on the side of justice and God will ultimately bring fairness to the world [6].

Underlying this idea that the world is moral is also a Tutuist moral theory.

From a Tutuist perspective, the highest good is the promotion of social harmony: ‘Harmony, friendliness, community are great goods.

Social harmony is for us the summum bonum—the greatest good’ [7].

This is an endorsement of the Afro-communitarian idea of Ubuntu, a concept that means ‘I am because you are and prescribes engaging in socially harmonious/positive communal relationships’.

Tutu’s quote is often interpreted as implying the idea that individuals ought to act in ways that promote the goods of others and, particularly, their welfare and virtue [8].

The prescription implies some duties.


Individuals are under a duty to promote the welfare and virtue of others, to the extent that it is reasonable, and it does not bring them great cost.

Moreover, if one is somehow responsible for other individuals’ goodness, refraining from aiding others may be a violation of the prescribed ethic [9].

For example, if I have a duty to take care of my children, refraining from promoting their welfare and virtue means that I fail to fulfill my duties.

Let us say that I refrain from taking my children to school, then I am violating a duty to promote their well-being.

In the African philosophical context, this duty to promote everyone’s well-being and virtue is something that all individuals are understood to have; but this universal rule does not have to be accepted for the purpose of this comment.

Instead, it is sufficient to accept that individuals have special duties towards significant others [10].

Parents, in particular, are in a unique relationship with their children and thereby may be the only ones realistically in a position to promote their welfare and virtue.


Why do we have duties towards some beings? Tutu’s own work has little to offer about normative ethics.

He routinely uses the concept of ‘human dignity’ to explain what is morally wrong with the Apartheid and racism [7][5][11].

Nonetheless, he not only never defines the concept, but the question at stake here is regarding non-human animal suffering, a topic which Tutu himself does not address.

Fortunately, several Tutuists have developed a Tutuist-inspired concept of moral status that can bring light to this question.

Grounded on Tutu’s idea that social harmony is the highest good, some Tutuist philosophers have argued that what makes beings morally worthy is the fact that they can either be the object or subject of communion.

That is, their capacity to either engage in social harmony or be a recipient of social harmony is what makes individuals deserve moral consideration [12][13].

Taking this on board, the most fundamental moral wrong involved in neglecting the welfare and virtue of animals is to fail to provide them what they are entitled to in virtue of their moral status [14].

A violation of dignity (human or not) is precisely a failure to treat individuals as they deserve [15].

According to this Tutuist understanding of the world, if Gasser were right that God does not rule the world according to moral rules, then God is not simply being amoral, He is being immoral.

For if the ethic prescribes that we are duty-bound to promote the welfare and virtue of others (especially significant others) when it is reasonable to do so, then a God who watches His creation suffer gratuitously without doing anything is like a father who neglects to take care of the welfare of his children.


In virtue of their moral status, animals are at the very minimum entitled to not be placed in situations where they suffer gratuitously.

For example, imagine an animal unknowingly drinks a poison I placed in front of him, and I happen to have the antidote in my pocket.

I can easily stop him from taking it or give him the antidote, but I just stand still watching the animal slowly dying.

My action is wrong in at least two ways.

I am guilty of misplacing the poison and should take responsibility; even if I were not guilty of it, it would be easy for me to stop it and I did not.

In both cases, it is clearly wrong for me to neglect the welfare of the animal.

The most fundamental reason underlying it is that in both cases I neglect that the animal has sufficient moral value to require any intervention and, thereby, neglect my duty towards the animal.

God, in the face of a situation which is not costly for Him and that He created, is duty-bound to address that evil, otherwise He is immoral.

Just like I could have avoided the suffering of the animal, so can God avoid gratuitous suffering and, to the extent that He does not, then He is being immoral.

The question becomes more pressing if I have special duties towards the suffering being.

Note that God as the creator is also the father of all existing beings and therefore has stronger duties towards them.

In other words, if God just watches an animal (which is His creation) suffering gratuitously when He could avoid it at a low cost, then He is disrespecting the moral status of this animal; God owes respect to the animal due to the animal’s moral status and has a special duty to honor this moral status because He is the father.


Gasser could object that this Tutuist theory does not have universal appeal and I need to make a stronger point with an example that is convincing not just to Tutuists but also other ethicists.

Hence, to understand the generally shared moral intuition underlying my argument, take another thought experiment which I contend to have wide appeal to individuals who hold different ethical views.

Peter Singer imagined a situation where a child has fallen into a pond and is drowning.

Someone passes by and sees that it would be fairly easy to pull the child out and the only cost to this person would be turning up late to work.

In such a situation, most would agree that there is an obligation to rescue the child because it is relatively easy to do this at a low cost without a significant burden [16].

Singer is a utilitarian, and he uses this argument to ground some utilitarian arguments about aid.

Nonetheless, the core point of Singer by formulating this thought experiment is not to show that utilitarianism is true.

Instead, his goal is to show that there is something most of us immediately recognize as morally wrong when confronted with this example.

Not aiding the child when the cost is so low is intuitively morally wrong, independently of one being a deontologist or a utilitarian.

The point I wish to make therefore is independent of what moral theory one prefers: without regard to whether one holds Tutuist ethics or a consequentialist perspective like Singer, one recognizes that the action is morally wrong.

In Gasser’s theory, God’s actions are analogous to the one of the person who lets the child die.

Given that God is omnipotent, it does not seem that God would endure great costs in avoiding animal suffering and it is Gasser’s burden to prove otherwise.


Moore’s thesis, if true, does, indeed, pose a problem to Christian ethics because Christian ethics is routinely grounded on metaphysical questions.

If the theory were true, then what God is like, how God is related to the world, what He did, what Jesus was like, and so forth seems to entail nothing regarding morality.

According to the naturalistic fallacy argument, there is no single value judgement entailed by these [23][24].

Nonetheless, I reject that this objection can challenge my argument.

As W.K. Frankena and Bernard Williams pointed out, even if one assumes that there is some kind of fallacy in the kind of reasoning that Moore is criticizing, it seems that Moore is being uncharitable towards most of his opponents [20][25].

The principle of charity requires that one’s statements are understood in the most reasonable or rational way possible [26][27].

Hence, when someone is stating, say, ‘X is pleasant, therefore X is good’ the most charitable interpretation of this is that this is an enthymeme where there is a suppressed premise ‘whatever is pleasant is good’ [21].

Moore is guilty of uncharitably neglecting that there is a hidden premise [20][25].


To show that I do not fall into a naturalistic fallacy, I wish to make explicit the chain of reasoning that led to my criticism.

The detailed reasoning of the current criticism is as follows: Syllogism 1 Social harmony is the highest moral good.

( p) Moral status is given in virtue of the capacity for engaging in social harmony either as a subject or an object.

( p) Morality prescribes (especially towards significant others) that one acts according to moral goods and that being’s moral status is respected.

( p) Therefore, morality prescribes one acts in socially harmonious ways and respects beings’ moral status (especially that of significant others).

( c) Syllogism 2 To respect a being’s moral status means to treat them with social harmony.

( p) Social Harmony is defined as the combination of solidarity and identification.

( p) Solidarity requires one to avoid evil and aid others when this is not unreasonably costly to do so. ( p) Therefore, one ought to avoid evil and aid others when this is not unreasonably costly.

( c) Syllogism 3 God does not avoid evils or aid others that He can reasonably avoid or aid those He can easily aid.

( p) Those who allow evils that can be reasonably avoided or who do not aid those they can easily aid are acting immorally. ( p) God is acting immorally. ( c) None of the premises above derives a moral proposition or a moral property from a natural proposition.

Instead, it looks at the situation and infers that there is an immoral act.

The inference from a specific situation is not a ‘naturalistic fallacy’.

The naturalistic fallacy is not that one cannot apply ethical principles to real circumstances [28].

Instead, it is that conclusions about value are inferred from premises about nature (rather than value statements).

Gasser could further challenge my contention on two grounds.

Firstly, Gasser could object that I am assuming God is a moral agent when the question being discussed is whether He really is a moral agent or not.

In other words, I am committing a petitio principii fallacy as I am assuming to be true without warrant what is to be proved, i.e., that God is a moral agent.

Secondly, Gasser can challenge my argument by contending that moral realism is false, and it is not verifiable.

Following an Ayerian line of thought, Gasser can uphold that there is nothing out there like ‘moral facts’ which can be observed.

According to this view, only those things that are empirically verifiable can be proved to be true.

Nevertheless, there is no such thing in the world that corresponds to a ‘moral fact’ [29].

Given that my theory seems to assume that there are moral facts out there, then my theory is false.

Having into consideration that this moral view of the world is incoherent, then the best alternative would be to uphold an amoral perspective of nature as Gasser does.


To answer the first objection, it is important to clarify what to be a moral agent means.

To be a moral agent is to have the capacity to discern right from wrong and thereby to be held accountable for one’s actions [30]. Is God a moral agent? He is necessarily so. If He is omnipotent, He must be a moral agent as if he were not, this would imply that he is not capable of understanding right and wrong and this is a contradiction in terms.

Put differently, the concept of ‘omnipotence’ necessarily entails that one is capable of exercising moral agency.

The same cannot be affirmed for immorality and amorality.

These refer to the absence of a capability to be moral or to a situation where morality is not a relevant factor (e.g., whether to drink jasmine or green tea) [9].

Thus, unless Gasser is willing to give up omnipotence, he cannot contend that God is not a moral agent.

Regarding the second objection, the first reply is that if this strong verificationist theory were true, this would render most universal propositions false.

For example, this would mean that propositions such as ‘all flamingos are pink’ or ‘all men are mortal’ are false because there is no possible way to verify all the cases.

Due to the fact that this theory is too demanding, generally speaking, philosophers consider today that it should be abandoned [18].

Instead, a better approach is to find theories that have a strong explanatory power.

Moreover, the fact that we have imperfect knowledge of moral facts does not stand as an argument against moral realism.

Natural sciences also revise their arguments and have imperfect knowledge and routinely revise their theories according to new paradigms [31].

We do not conclude from this that there are no natural facts and there is no reason to infer differently regarding moral facts.


A second possible interpretation of Gasser’s argument is that it is not that the world is amoral but that divine and human moral principles are distinct.

This interpretation is suggested in the following passages: ‘If God is in no way dependent on the world and cannot be influenced by it, the moral standards obvious to us are hardly applicable to God.

Since the problem of evil lives from these standards, evils in the world no longer count as direct counter-evidence to God’s existence as it might be the case that our moral concerns are in no way God’s concerns.

’ [ 1] And: ‘God is responsive to values, but these values may not make an exclusive reference to us.’ [ 1] According to this view, creation is moral but not human-driven.

So, according to this interpretation, it is not that God is amoral like in the previous case.

Instead, His moral code is different and not responsive to humans; thereby He cannot be judged by such standards.

In this second interpretation, Gasser is not concerned about the naturalistic fallacy given that he himself seems to admit that God is the ultimate source of morality, which is precisely the kind of problem that proponents of the naturalistic fallacy are at odds with.


The first problem with this view is that it collapses into one of the interpretations of the Book of Job that Gasser finds incomplete and unconvincing.

If the view that moral standards do not apply to God is true, in the face of such horrendous injustices as Job seems to suffer, we can only conclude that God’s moral rules are beyond human understanding and that Job should simply accept his destiny and humbly submit to God in the face of His greatness. But if there is an inscrutable moral code for humans, then this has several negative implications which seem not only to be at odds with Christian morality but also to be morally questionable more generally.

If this theory were true, this would mean that humans can never be held accountable for moral wrongs.

Just like a baby who cannot understand what is right and wrong and should not be held morally responsible for her actions, humans likewise should not, in this view, be held responsible for not following a rule which is impossible to understand: their relationship to God’s moral code is analogous to the one of babies vis à vis the moral code of adults.

Gasser may reply that there is a human moral code given by God which humans should humbly follow, and therefore they should be held accountable for not following it even if they do not understand it.

Nonetheless, the existence of a general moral code which is simply repeated rather than understood brings further problems.

No general moral code can be of any use for particular situations unless it is understood and, thereby, applied in particular cases.

In fact, a salient Christian idea is that the Bible needs to be understood as a whole so that its values are honoured.

According to this view, without such understanding, it is not possible to find moral guidance in the Bible [32].

Furthermore, it seems that a necessary requirement for one to be a moral person is that one understands and, thereby, can reasonably predict the consequences of one’s actions.

Without knowing these, one’s actions are simply random acts or repeated meaningless forms of behaviour [18].

There is little or nothing praiseworthy about a robot mechanically repeating an action (e.g., a traffic sign changing colours to regulate the traffic).

What is praiseworthy is that a certain person knows she is doing something that can achieve a greater good or that honours a certain value.

Even if there is some value in repeating a morally right action, it is surely better to do something understanding it is morally right than to simply blindly and mechanically do it.

Again, this coheres with the salient Augustinian Christian tradition of thought where moral actions are better if they are understood [33].

More intriguingly, if God does not provide an understandable moral code for humans, then He would be acting immorally.

As explained in the previous section, under Tutuist ethics, everyone (including God) is duty-bound to help individuals acquire virtue. But an omnipotent being not creating an understandable moral code means basically to create/allow unnecessary confusion amongst humans who will misunderstand His moral code and, thereby, cause evil.

If this were true, then God would be responsible for the moral evils in the world because He had provided misleading guidance to humans.

For example, in a war situation, a general may be responsible for a war crime committed by her soldiers if she has deliberately misled them to believe, say, that innocent civilians were soldiers and this led her soldiers to kill innocents.

To put it in a syllogism: Social harmony requires one to help others to act morally if costs to oneself are not unreasonable.

( p) When one fails to aid others to be morally better or causes others to be worse, one is acting immorally.

( p) There are occasions where God both fails to aid individuals and leads individuals to act immorally.

( p) Therefore, God is immoral. ( c) Note that the point is not that God should reveal Himself.

As Swinburne suggests, there may be plausible reasons for God to not be obviously present as a stimulation for humans to do good [18][19].

Instead, the point is that if it is impossible for humans to know God, then Him not revealing moral norms cannot be explained as a method for stimulating humans to do good, but as undermining the possibility of doing good.


In this piece, I evaluated Gasser’s argument for the problem of animal suffering considering Tutu’s thought. I argued that Gasser’s view has unacceptable implications for Christian doctrine, i.e., that God is immoral.

Hence, Gasser’s theory, if one starts from a Christian viewpoint, cannot be true.

Moreover, I argued that Gasser’s views also imply that humans can neither be praised nor blamed for their moral choices and actions.

Finally, I contended that holding this moral perspective about God does not imply the naturalistic fallacy.

Nonetheless, this does not disprove the existence of God because not only are there other theodicies which do not face the same problem, but also there are other concepts of God outside Christianity that do not understand God as morally perfect.

Further research should explore a Tutuist explanation of the problem of animal suffering.


In this reply, I aim to clarify my ideas presented in a recent paper and to address criticisms that have been raised by Luis Cordeiro-Rodrigues regarding my interpretation of (animal) suffering and God.

Critical objections are a welcome means of removing ambiguities or clarifying implicit assumptions.

For this reason, I am very grateful that Luis Cordeiro-Rodrigues has written a reply to my paper “Animal Suffering, God and Lessons from the Book of Job” [1].

This gives me the opportunity to rethink this topic again and try to present it in a more precise manner.

Central to the problem of evil is the presupposition that God is a morally perfect being.

The core of the discussion works with the following principles: (a) A morally perfect being eliminates evil as far as it is possible.

( b) Within the realm of logical possibilities, an omnipotent being has no limits of agency.

With these two principles at hand, one can argue that we should expect that a morally perfect and omnipotent being will eliminate all evils unless it is impossible to do so. Since there are many evils in the world that we are certain it is logically possible to avoid or eliminate, we have very strong evidence that such a being does not exist.

Advocates of theism have mainly responded to this challenge by pointing out that there might exist possible higher goods (e.g., freedom of will, character building, union with God, etc.), which can only be achieved by allowing evils in the world [2].

As a consequence, God is morally justified in permitting these evils as they are necessary and indispensable instruments for bringing about higher goods.


Once this instrumental relation between evils and higher goods is not exclusively reserved for human beings but applied to creation in general, then also the suffering of animals can be explained in a similar fashion.

Trent Dougherty, for example, develops an expanded soul-making theodicy for animals: Accordingly, God, by being in a caring and loving relationship with all of creation, will enfold the suffering of any creature in a greater good that organically defeats this evil [3].

In a recent paper [1], I argue that this understanding of God is couched in overly personalistic terms.

God is imagined similar to a human person and judged accordingly with the moral standards appropriate for human agents.

Without doubt, there is this tradition of a personal conception of God; however, it should not be ignored that there is also another powerful tradition, namely that of so-called classical theism, in which personal attributes of God are of secondary importance.

This is so because God as creator is not a part of the created world, and therefore God cannot belong to the moral community as humans do.

A main problem of the problem of evil is the anthropomorphic understanding that the human–human relationship is the template for all kinds of relationships, including the divine–human one.

Once this view is rejected, the problem of evil changes as the central moral assumptions regarding God ought to be revised.

We cannot apply our moral categories to God as these categories belong to the created but not to the transcendent order of being.

The idea I am proposing is that God is interested in its creation, but the essential values that shape the divine view are to be explicated less in terms of moral categories than in terms of non-moral goods such as complexity, fecundity, creativity, adaptability, or the will to live.

To put it more pointedly, humans might be less central to God’s overall creational purposes than personalistic interpretations of a theistic God generally presuppose.

Such a view may seem odd at first glance, but—according to my argumentation, which draws also upon exegetical interpretations such as those of Carol A. Newsom [4] and Steven Chase [5]—the divine speeches in the Book of Job can be understood in precisely this way: God teaches Job to expand his focus by opening up the narrow circle of human-oriented moral questions toward the astonishing and mysterious sublimity of natural and cosmic processes where moral categories are non-applicable.


At this point, it is important to emphasize that I do not defend an ananthropocentric view, that is, the view that humans are completely irrelevant to the divine purpose and do not matter to God at all [6].

Rather, humans are one aspect of God’s perspective on creation among those others.

We are one but not the only essential part of the creational picture.

Accordingly, the divine perspective is related to creation in a twofold way: One perspective understands the human being as a part of the larger universe in which moral categories have no direct point of reference.

The other perspective, however, is related to the individual human being as a direct addressee of God as the divine speeches in the book of Job illustrate and, thus, also involves the moral order intrinsically connected to the human life-form.

Both perspectives are separated by the moral sphere as it distinguishes the human and the non-human realm: Responsibility, justice, empathy, or orientation toward moral values are essential coordinates of the human world.

These features constitute the human life-form in the first place.

When it comes to the world of other living beings or the non-living dimensions of creation, however, these features have no essential role to play.

Most scientists consider animals to be a-moral because they lack the cognitive capacities required for (full) moral agency [7].

Accordingly, we do not blame a predator for killing a prey because hunting and killing animals is a central feature of a predator’s life-form, but moral understanding is not.

In the light of this understanding, the divine speeches in the Book of Job highlight both perspectives and the tension that comes with them.

Newsom writes: “What Job has just heard in the divine speeches, however, is a devastating undermining of his understanding of the unproblematic moral continuity between himself, the world, and God. It is a profound loss of unity, a recognition of the deeply fractured nature of reality” [4].

There is not one perspective from which the world is grasped as unified and well-ordered but a holistic and an individual perspective, each of which is shaped by different normative schemes.

To a structurally similar conclusion comes philosopher Wes Morriston: “The Hassidic teacher, Rabbi Bunam, said that ‘A man should carry two stones in his pocket.

On one should be inscribed, “I am just dust and ashes”.

On the other, “For my sake was the world created”. And he should use each stone as he needs it.’ The experience of the whirlwind has taught Job to use the first stone. But what we need, and what the book of Job tries, with only partial success, to teach us, is how to use them both together” [8].

Both Newsom and Morriston point out that the tension in such an understanding of the God–world relationship cannot be brought to a higher synthesis; rather, it is our lot to endure it.

In his encounter with the divine, Job realizes this essential feature of the world and our tragic place in it.


X-linked adrenoleukodystrophy is a peroxisomal disorder caused by mutations in the ABCD1 gene (http://www.x-ald.nl) (Mosser et al., 1993), which codes for the transporter of very long-chain fatty acids (5C22:0) (van Roermund et al., 2008).

ABCD1 defi- ciency impairs peroxisomal very long-chain fatty acid degradation resulting in increased cytosolic very long-chain fatty acid-CoA levels (Kemp et al., 2011), which are further elongated by the very long-chain fatty acid-specific elongase, ELOVL1 (Ofman et al., 2010), thus causing very long-chain fatty acid accumulation (Moser et al., 1981; Kemp and Wanders, 2010).


In males, disease course and symptomatology have been studied extensively (Engelen et al., 2012).

The clinical spectrum ranges from isolated adrenocortical insufficiency, slowly progressive myelopathy and peripheral neuropathy in adulthood (adrenomyeloneuropathy) to a rapidly progressive and fatal cerebral demyelinating disease in boys or adult men (cerebral-adrenoleukodystrophy).

The most frequent phenotype in males is adrenomyeloneuropathy (van Geel et al., 1994; Engelen et al., 2012).


There have been several studies and observations concerning the phenotype of X-linked adrenoleukodystrophy carriers.

O'Neill et al. ( 1984) described 21 obligate carriers and several were shown to have signs and symptoms of myelopathy.

It was esti- mated that 50% develop symptoms at some point in their life (Moser et al., 1991).

In a small study, eight females were found to have myelopathy and in a retrospective study the frequency of symptoms increased with age (van Geel, 2000; Schmidt et al., 2001).

These studies provide no information on the prevalence of neurological symptoms in the entire population of X-linked adrenoleukodystrophy carriers, as data were acquired in small groups and may be biased towards females with symptoms.


Adrenal insufficiency is rare in X-linked adrenoleukodystrophy car- riers (el-Deiry et al., 1997), and there are only a few reports of X-linked adrenoleukodystrophy carriers that developed cerebral- adrenoleukodystrophy (Pilz and Schiener, 1973; Jung et al., 2007).

Some studies suggested that disease severity could be related to the pattern of X-inactivation, whereas others refuted this observation (Migeon et al., 1981; Watkiss et al., 1993; Maier et al., 2002; Salsano et al., 2012).


The primary goal of our study was to estimate the proportion of X-linked adrenoleukodystrophy carriers that develop symptoms and to characterize these symptoms both clinically and with ancil- lary investigations.

A secondary goal was to determine if the X-inactivation pattern of the ABCD1 gene was associated with symptomatic status.


We designed a prospective cross-sectional cohort study.

Female car- riers over the age of 18 years were eligible to participate.

They were recruited from the outpatient clinics of the Academic Medical Centre and the Medical Centre Alkmaar from 2008-10.

To prevent bias for symptomatic women, we tried to examine all female relatives with X-linked adrenoleukodystrophy of the women participating.

Through the Dutch X-linked adrenoleukodystrophy patient organization, letters to invite X-linked adrenoleukodystrophy carriers to participate were sent to all members.

The study protocol was approved by the local Institutional Review Board.

After informed consent was obtained, women visited the outpatient clinic for assessment, which included questionnaires, neurological examination, neurophysiological tests, blood samples and skin biopsy.


A careful history with emphasis on neurological complaints and neuro- logical examination was performed.

In particular, symptoms of incon- tinence, gait disorder, maximum walking distance, and sensory disturbance were recorded.

Urinary incontinence was defined as urge incontinence.

Stress incontinence was not considered a symptom of myelopathy.

A gait disorder was considered present if the walking distance was significantly reduced or running was not possible.

Sensory complaints were recorded if there were paraesthesias or numbness in the lower extremities.

Sensory disturbances on examin- ation were considered present if there was reduced sensation to touch, pinprick, vibration or position sense in the lower extremities.

Muscle strength was rated using the Medical Research Council Scale and spasticity was rated using the Ashworth scale (Ashworth, 1964).


An Expanded Disability Status Scale value was scored based on the history and physical examination performed during the visit and recorded in the medical chart (Kurtzke, 1983).


Carriers with peripheral neuropathy or myelopathy were considered symptomatic.

Myelopathy was considered present if: (i) there were symptoms of myelopathy (for instance sphincter disturbance); and (ii) signs of myelopathy on neurological examination (pyramidal tract or dorsal column signs) were present.

If only symptoms were present (for instance faecal incontinence) but no signs on neurological exam- ination, myelopathy was not scored as present.

Peripheral neuropathy was considered present if nerve conduction studies and/or EMG were abnormal (as described below).


All participants completed the Dutch version of the SF-36 (Quality of Life Assessment) (Aaronson et al., 1998), and the AMC Linear Disability Scale (Weisscher et al., 2007).

SF-36 values can be com- pared to reference values for the Dutch population, matched for gender and age.

The AMC Linear Disability Scale values were


Nerve conduction studies and EMG were performed according to a fixed protocol, allowing comparison with reference values and possibly follow-up in the future (Verhamme et al., 2009).

Compound muscle action potential amplitudes, baseline to negative peak, were recorded from the abductor pollicis brevis, abductor hallucis brevis, and extensor digitorum brevis on one side.

A summated compound muscle action potential amplitude was calculated.


Motor nerve conduction velocities were calculated after distal and proximal stimulation of the right median, tibial, and peroneal nerves.

Sensory nerve conduction velocity, calculated based on negative peak markers, and sensory nerve action potentials amplitudes, baseline to negative peak, were investigated for the left median nerve and right sural nerve.

A summated sensory nerve action potential amplitude was calculated.


Electromyography was done of the left anterior tibial, and interosseus I/II muscles.

An axonal peripheral neuropathy was diagnosed if in two separate nerves at least one parameter was outside the 95% confidence interval (CI) and the criteria for demyelinating peripheral neuropathy were not ful- filled (Van Asseldonk et al., 2005).

Somatosensory evoked potentials of left median and the posterior tibial nerves on both sides and brain- stem auditory evoked potentials on both sides were registered, con- forming to local protocols (Aramideh et al., 1992; Aalfs et al., 1993).


Venous blood samples were taken and plasma and lymphocytes stored as described previously (Engelen et al., 2010).


From skin biopsy material primary fibroblast cell lines were generated and cultured as described (Valianpour et al., 2003).

Very long-chain fatty acids (C26:0 and the C26:0/C22:0 ratio) were determined in plasma and fibroblasts as described (Valianpour et al., 2003).


Peroxisomal beta-oxidation activity was measured essentially as described by incubating cells with 30 mM deuterium-labelled C22:0 (D 3 -C22:0) (Kemp et al., 2004).

ABCD1 protein levels were deter- mined by immunofluorescence and quantitative immunoblot (Kemp et al., 1996; Zhang et al., 2011).


Mutation analysis of the ABCD1 gene was performed as described (Boehm et al., 1999).

ABCD1 allele-specific expression (ABCD1 allele-specific expression) of 38 carriers was measured by pyrosequen- cing.

RNA was extracted from primary fibroblasts and complementary DNA synthesized as described (Bieche et al., 2001).

The ratio of normal versus mutant allele expressed was measured by pyrosequen- cing.


Complementary DNA was PCR-amplified using biotin-labelled primers followed by quantitative pyrosequencing using a PyroMark Q96 ID instrument (Qiagen).

Biotin-labelled single-stranded amplicons were isolated following the protocol using the Qiagen PyroMark Q96 Workstation pyrosequenced with a sequencing primer (PCR- and sequencing-primers are provided in Supplementary Table 1).

Each sample was pyrosequenced in the upper direction (with lower biotin- labelled amplicons) and in lower direction (with upper biotin-labelled amplicons).

The ratio of normal versus mutant allele was measured using the PyroMark ID software (Qiagen).


For each carrier, the ABCD1 allele-specific expression value represents the mean of upper and lower reads (determined in two independent experiments).

For included samples, variations between upper and lower reads were 410%.

For validation, we repeated the experiment for a subset of samples (n = 6) using different PCR primers or independent comple- mentary DNA samples.

The ABCD1 allele-specific expression values were similar (variations 44%).


ABCD1 allele-specific expression pattern was defined as 'severely' skewed with an expression of the normal allele 410% and 'moder- ately' skewed with an expression of the normal allele between 11-25%, similar to a previous report (Miozzo et al., 2007).

In the manuscript ABCD1 allele-specific expression will be referred to as X-inactivation.


For five patients the ABCD1 allele-specific expression analysis was not possible because of the complexity of the mutation.

Instead, ABCD1 allele-specific expression values were calculated using ABCD1 protein immunofluorescence and determination of the ratio of ABCD1 protein positive and negative cells.


Data were analysed with IBM SPSS statistics version 19 (IBM Inc.), Microsoft Excel from Office 2010 and Prism version 5 (GraphPad Software).

Depending on the distributional properties, outcome meas- ures were expressed as means AE standard deviation (SD) or as medians with ranges.

Statistical significance was assessed by independent sample Student's t-test for normally distributed continuous data and the Wilcoxon test for non-normally distributed continuous data.

All reported P-values are two-sided and were not adjusted for multiple testing.

The SF-36 scores were converted to z-scores based on the Dutch norm population, using the appropriate age groups.

AMC Linear Disability Scale scores were analysed as described previously (Weisscher et al., 2007).

The relationship between symptomatic status, age and the pattern of X-inactivation in skin fibroblasts were analysed by logistic regression.


Forty-six females from 26 kindreds were enrolled in the study (one kindred with five females, two with four females, two with three, five with two, and 17 with one).

Twelve were recruited through the outpatient clinic, 34 were relatives of these females or responded to the letter sent to members of the Dutch X-linked adrenoleukodystrophy patient organization.

Complete clinical and electrophysiological data were available from 46 females, blood samples were available from 45 females (one refused venepunc- ture) and skin biopsies were obtained from 43 women (two refused skin biopsy, one fibroblast culture failed because of a yeast infection).

The age of the included females ranged from 22 to 76 years (average 48 AE 13 years) (Fig. 1).

Mutation analysis was done in 45 females.

For one obligate carrier no leucocytes were available, the mutation in that subject was considered to be the mutation found in her father and paternal uncle (Table 1).


Symptoms, signs and Expanded Disability Status Scale scores are presented in Table 1 and summarized by age group in Table 2.

Complaints of incontinence (both urinary and faecal), abnormal gait, and sensory symptoms were common.

Myelopathy was found in 29/46 (63%) and peripheral neuropathy in 26/46 (57%).

Two of 46 (4%) had peripheral neuropathy without myel- opathy, and 11/46 (24%) had myelopathy without peripheral neuropathy.

Thirty-one of 46 (67%) were considered symptomatic because of either myelopathy and/or peripheral neuropathy.

The percentage of women with signs and symptoms suggestive of myelopathy increased with age (Table 2).

The percentage of symptomatic females increased from 2/11 (18% in the youngest age group) to 7/8 (88% in the oldest age group).

Logistic regres- sion showed a significant relationship between age and symptom- atic status, with the probability of being symptomatic increasing clearly with age [P = 0.002; odds ratio (OR) 1.2 (1.07-1.34); Fig. 2].


Quality of life, as measured by the SF-36, was not statistically significant different between asymptomatic and symptomatic car- riers.

However, on measures that are related to physical disability (Fig. 3A), there was a trend towards a statistically significant dif- ference.

There were no differences in emotional or mental health between asymptomatic and symptomatic carriers.

The levels of disability were measured by the AMC Linear Disability Scale scores (Fig. 3B) and these were compared to data from the CARPA study, a prospective cohort study in Parkinson's disease (Post et al., 2011).

The AMC Linear Disability Scale scores were lower in the group of symptomatic X-linked adrenoleukodystrophy carriers compared with the asymptomatic carriers.

Symptomatic carriers had AMC Linear Disability Scale scores comparable to patients with Parkinson's disease at baseline and 1 year after diagnosis.


Of the X-linked adrenoleukodystrophy carriers 26/46 (57%) had a peripheral neuropathy.

The abnormalities mostly consisted of a reduction in compound muscle and sensory nerve action potential amplitudes, with a marginal slowing in nerve conduction velocity (Table 3).

This is consistent with a sensorimotor axonal peripheral polyneuropathy, as has been reported to occur in males with X-linked adrenoleukodystrophy (van Geel et al., 1996).


The somatosensory evoked potential from the median (arm) nerve was abnormal in 3/42 (7%) of X-linked adrenoleukodystrophy carriers (four registrations failed because of technical reasons), of the tibial (leg) nerve 14/44 (30%) were abnormal (two registra- tions failed) (Table 4).

The median nerve somatosensory evoked potential was normal in all 15 asymptomatic carriers.

The posterior tibial nerve somatosensory evoked potential was abnormal in one (7%) asymptomatic carrier.

Three of 27 (11%) symptomatic carriers had an abnormal median nerve somatosensory evoked potential and 13/29 (45%) had an abnormal tibial nerve somato- sensory evoked potential.

The average latency of the cortical peak increased with age for median nerve and tibial nerve somatosen- sory evoked potential.

The percentage of abnormal somatosensory evoked potentials increased with age (Table 4).


Brainstem auditory evoked potential measurement failed for tech- nical reasons in one participant (Table 4).

For the entire group, 26/45 (58%) had an abnormal brainstem auditory evoked poten- tial, mostly consisting of an increased I-V and I-III interval.

In the asymptomatic group, 3/13 (23%) had a normal brainstem audi- tory evoked potential.

In the symptomatic group this proportion was 23/32 (72%) had an abnormal brainstem auditory evoked potential.

There were no significant left-right differences.


Plasma C26:0 were increased (2.26 AE 0.69 mmol/l; reference values 1.05 AE 0.078 mmol/l; Table 5).

Thirty-one of 45 (69%) had abnormal plasma very long-chain fatty acids levels.

There were no significant differences in plasma C26:0 levels between asymptomatic and symptomatic carriers (P = 0.16).

In contrast to what we reported before, C26:0 levels did not increase with age (Stradomska and Tylki-Szymanska, 2001).


Fibroblast C26:0 levels were increased in 37/43 (86%), and the C26:0/C22:0 ratio in 36/43 (84%) (Table 5).

The D 3 -C16:0/D 3 - C22:0 flux-ratio was decreased in 26/43 (60%), indicating reduced peroxisomal very long-chain fatty acids beta-oxidation capacity.

Two of 43 (5%) carriers had normal C26:0 levels in plasma and fibroblasts, and 1/45 (2%) had normal C26:0 levels and normal peroxisomal beta-oxidation.

There was a clear correl- ation between the ABCD1 protein levels in fibroblasts and the residual peroxisomal beta-oxidation capacity, C26:0 synthesis and very long-chain fatty acids levels (Fig. 4).


The distribution of ABCD1 allele-specific expression (which will be referred to as the pattern of X-inactivation) is shown in Fig. 5A.

There was no evidence for skewing to either the mutated or normal ABCD1 allele.

The pattern appeared random and the median value was exactly 0.5.

The pattern of X-inactivation was similar between age groups (Fig. 5B).

This contrasts with previous reports that documented a highly skewed pattern of X-inactivation in X-linked adrenoleukodystrophy carriers (Migeon et al., 1981).


We subsequently correlated the pattern of X-inactivation with biochemical parameters in fibroblasts, i.e. ABCD1 protein levels, residual beta-oxidation activity and very long-chain fatty acids levels (Fig. 6).

A clear correlation was found between the pattern of X-inactivation and the biochemical parameters in the fibro- blasts.

Indeed, if there was preferential expression of the normal allele, the level of ABCD1 protein was higher, residual rates of beta-oxidation were higher, very long-chain fatty acids synthesis was lower, and very long-chain fatty acids levels were lower, and vice versa.


To determine if the pattern of X-inactivation correlates with symptomatic status it is important to consider age as there is a strong correlation between age and symptomatic status (Fig. 2).

Logistic regression showed no correlation between symptomatic status and the ABCD1 allele-specific expression pattern, also after adjustment for age [P = 0.74; OR 1.00 (0.97-1.02)].

In Fig. 5C and D the distribution of X-inactivation in asymptomatic and symptomatic carriers is plotted.


X-linked adrenoleukodystrophy carriers develop neurological symptoms.

In fact, one of the earliest descriptions of an adreno- myeloneuropathy-like phenotype in X-linked adrenoleukodystro- phy was a female patient (Penman, 1960).

This is not common knowledge among physicians (Jangouk et al., 2012).

Several X-linked adrenoleukodystrophy carriers were reported to undergo cervical laminectomy for suspected cervical spondylogenic myelop- athy (van Geel et al., 1997).


In this largest prospective cross-sectional cohort of female carriers to date, we found that neurological abnormalities are common and that the frequency increases steeply with age.

The main symptoms were consistent with myelopathy, as in males with adrenomyeloneuropathy.

It is striking how often faecal incontin- ence is reported as an early symptom.

One should bear in mind, however, that it is often not voluntarily reported because it is felt to be embarrassing.

To our knowledge, this is not reported often in males with adrenomyeloneuropathy or in other degenerative myelopathies, for instance, hereditary spastic paraplegia, albeit that this feature may not have been specifically asked for (Salinas et al., 2008).