"DNA methylation data from neonatal blood spots can be used to accurately predict age and maternal smoking status"

There were two primary reasons why I wanted to blog about the findings reported by Eilis Hannon and colleagues [1]: first, the focus of the study was "to identify DNA methylation biomarkers of ASD [autism spectrum disorder] detectable at birth", and second, the authors actually "identified robust epigenetic signatures of gestational age and prenatal tobacco exposure, confirming the utility of DNA methylation data generated from neonatal blood spots."

The first reason, looking at DNA methylation biomarkers in relation to autism, follows other similar initiatives down the years (see here for example) on the back of some significant interest in how "epigenetic variation induced by non-genetic exposures" might complement/fill in some gaps left by more traditional genetic studies. Epigenetics by the way, seemingly means different things to different people, but is currently summarised as "the study of heritable changes in gene function that do not involve changes in the DNA sequence." DNA methylation reflects one epigenetic process (there are others). The second reason - robust epigenetic signatures linked to gestational age and prenatal tobacco exposure - actually turned out to be the more important finding, or at least the more significant finding, reported by Hannon et al hence the title of this post...

'Guthrie cards' are mentioned as the starting material for the Hannon paper, and yet another hat-tip to a true medical pioneer, Robert Guthrie (and his team), who has saved multitudes of lives with his cards used to collect and store neonatal blood spots. As well as being used to screen for various potential inborn errors of metabolism (some of which seem to have something of a relationship with some autism), those archived blood spot cards have also proved to be important research fodder too (see here).

Hannon garnered neonatal methylomic data for approaching 1300 individual - "comprising equal numbers of ASD cases and matched controls, 50% male/female" - derived from "the iPSYCH case–control sample" based in Denmark. We are told that: "DNA methylation was quantified across the genome using the Infinium HumanMethylation450k array" bearing in mind this technique/system "only assays ~ 3% of CpG sites in the genome." Alongside: "Matched genome-wide single nucleotide polymorphism (SNP) genotyping data from the same individuals enabled us to undertake an integrated genetic–epigenetic analysis of ASD, exploring the extent to which neonatal methylomic variation at birth is associated with elevated polygenic burden for ASD."

Results: with regards to a neonatal methylomic 'signature' for autism or ASD, nothing significant was detected. Obviously one has to bear in mind the limitations of the assay/method used and the fact that blood from bloodspots represents only one type of tissue (DNA methylation patterns are not necessarily the same across different tissues). I was also interested to see the authors talk about "the chronology of sample collection prior to ASD diagnosis" and the 'plausability' that they were "looking too early on in the disease process." No, autism isn't 'a disease', but this work might provide some support for the idea that the processes and onset of autism is not always set either during conception nor during gestation (see here). Dangerous thinking for some people of a sweeping generalisation ilk...

Having said all that, researchers did talk about a "significant association between increased polygenic burden for autism and methylomic variation at specific loci" but I'd like to see replication of this effect before any big claims are made.

Then to those other findings of "robust epigenetic signatures of gestational age and prenatal tobacco exposure" and what that could mean to several different areas of research, autism and beyond. I'm a little surprised that the authors didn't make more of their 'robust findings' in their discussion of these results. I appreciate that their primary aim to "identify DNA methylation biomarkers of ASD detectable at birth" was not met with startling success but the suggestion of a tell-tale epigenetic sign of exposure to maternal smoking during pregnancy for example is, I would have thought, an important advance. Certainly one that could be at least relevant to various other studies, including those related to an important comorbidity that seems to be 'over-represented' in relation to some autism (see here) and perhaps more.

To close, it's not the first time that the Star Wars universe has been subject to peer-reviewed 'science' but the paper by Hatters Friedman and colleagues is an interesting one...

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[1] Hannon E. et al. Elevated polygenic burden for autism is associated with differential DNA methylation at birth. Genome Med. 2018 Mar 28;10(1):19.

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"findings suggest a protective effect of CRP" on schizophrenia risk?

Science is often a puzzling endeavour. Sometimes, just when you think that you've got something nailed down, scientific results appear that 'trash' long held, cherished beliefs. So it was with the publication of the results by Fernando Pires Hartwig and colleagues [1] who presented findings looking at "the effect of inflammatory markers on schizophrenia risk" based on the use of "a mendelian randomization (MR) design."

MR, by the way, is a interesting technique based on the principle that "genetic variants that either alter the level of, or mirror the biological effects of, a modifiable environmental exposure that itself alters disease risk should be related to disease risk to the extent predicted by their influence on exposure to the environmental risk factor." It's a technique that has already been applied to inflammatory markers in the context of schizophrenia on more than one occasion (see here and see here). Those inflammatory markers studied have included ones which were covered by the Hartwig paper. Specifically: "Genetically elevated circulating levels of C-reactive protein (CRP), interleukin-1 receptor antagonist (IL-1Ra), and soluble interleukin-6 receptor (sIL-6R)." I should also point out that Hartwig and colleagues have some research form in the area of applying MR to various aspects of medical science (see here).

As per an accompanying editorial on the Hartwig paper [2], the long-and-short of it was that researchers "used 2-sample MR to test for a potentially causal relationship between inflammation and schizophrenia and to improve inference for the association between genes, inflammatory biomarkers, and risk of developing schizophrenia." I can't claim any specific expertise in the use of MR (see here for a good overview [3]) but it appears that data on single-nucleotide polymorphisms (SNPs) in relation to those inflammatory markers was used to test whether said markers might be linked 'causally' to risk of schizophrenia. Their results were interesting: "we did not find strong evidence that lifelong exposure to increased action of these proinflammatory cytokines increases schizophrenia risk, as previously hypothesized" and indeed that: "blockade of IL-6 effects and low CRP levels might instead increase schizophrenia risk." This is contrary to quite a lot of other research in this area (see here for example).

There are a few words of caution to attach to the Hartwig results that need mentioning not least the primary tenet on which analyses are based: genetic variants (SNPs) affecting something like CRP are of primary importance to schizophrenia. I don't for example, see anything in the data looking at gene function/expression being affected as a result of non-structural changes to the genome via something like epigenetic 'alterations' for example (and there is such a thing as epigenetic Mendelian randomization y'know). I say this on the basis that other genes involved potentially involved in processes linked to DNA methylation have also been *associated* with cases of schizophrenia (see here). The authors also caution that their analyses are based on "lifelong exposure to elevated cytokine and CRP levels" and that exposure during 'critical windows' might be the important issue when it comes to any change in schizophrenia risk. Similarly they note that "it is possible that IL-6 and CRP effects on schizophrenia risk are related to a maternal effect (eg, maternal susceptibility to infections during pregnancy), so that our findings are explained by the correlation between maternal and offspring genotypes." This final point is based on the idea that maternal infection during pregnancy (or the biological consequences of) seems to be quite a big risk factor for at least some presentations of schizophrenia (see here) as well as [cautiously] other labels (see here). Here, the importance of a reprogrammed immune system during pregnancy might also come into play alongside any maternal 'susceptibility'.

Personally I'm not yet ready to totally trash the idea that the immune system, and specifically elevations in inflammatory markers such as CRP and other pentraxins, might not be important to some schizophrenia risk in a more detrimental way. I appreciate that one has to be careful when talking about immune system markers and their inflammatory direction (see here for some chatter on IL-6 and its pro- and anti-inflammatory natures) but the existing data is too evident to just discard on the basis of one new study, despite it's scientific prowess. I don't however doubt that there may be several confounding variables linked to increases in CRP in schizophrenia; not least the impact of something like increased body mass index (BMI) that seems to follow some cases of schizophrenia [4]. These variables need to be further explored, particularly in the context of what side-effects pharmacological management of schizophrenia might have (see here). And I also hat-tip the paper by Manu and colleagues [5] applying the Bradford Hill's guidelines on 'causation' to this area and concluding that (upto 2014) "there is insufficient evidence that the replicated, strong association between schizophrenia and elevated inflammatory markers has etiopathological relevance"...

For now however, the Hartwig findings reiterate that science is an ever-changing, ever-evolving process...

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[1] Hartwig FP. et al. Inflammatory Biomarkers and Risk of Schizophrenia: A 2-Sample Mendelian Randomization Study. JAMA Psychiatry. 2017. Nov 1.

[2] Byrne E. et al. Inference in Psychiatry via 2-Sample Mendelian Randomization—From Association to Causal Pathway? JAMA Psychiatry. 2017. Nov 1.

[3] Sheehan N. et al. Mendelian Randomisation and Causal Inference in Observational Epidemiology. PLoS Med. 2008; 5(8): e177.

[4] Fernandes BS. et al. C-reactive protein is increased in schizophrenia but is not altered by antipsychotics: meta-analysis and implications. Mol Psychiatry. 2016 Apr;21(4):554-64.

[5] Manu P. et al. Markers of inflammation in schizophrenia: association vs. causation. World Psychiatry. 2014 Jun; 13(2): 189–192.

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L-methylfolate administration and autism: a case report

I should have really titled this post 'another case report' given yesterday's entry on this blog talking about a case of [untreated] PKU and autistic behaviours/diagnosis. Here I am again talking about another N=1 with autism in mind and specifically the findings reported by Kim Siscoe & David Lohr [1] on how: "L-methylfolate supplementation improved symptoms of aggression and disruptive behavior in a child with autism who tested positive for the C677TT allele of the methyltetrahydrofolate reductase enzyme gene."

First things first. This was a case report; please keep that in mind. Second, I am not a medical doctor and don't provide medical or clinical advice on this blog. Within those caveats I am however very interested in the Siscoe/Lohr observations.

Why? Well, methylene tetrahydrofolate reductase (MTHFR) (gene and enzyme) has featured quite a bit on this blog in light of findings linking gene and enzyme to cases of autism (see here and see here for examples). The idea is that MTHFR serves a primary function in reducing the compound 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-methyltetrahydrofolate - another name for L-methylfolate -  the reduced and methylated form of folic acid, is an important methyl group donor for the recycling of homocysteine back to methionine utilising vitamin B12 along the way (see here for a nice hand drawn graphic). The implications of disruptions to MTHFR (gene and enzyme) are potentially multiple but include effects on methyl group donor ability (methyl groups potentially linked to things like DNA methylation as part of all that epigenetics jazz that you hear so much about these days) and effects on downstream metabolites such as those related to homocysteine metabolism (see here).

So Siscoe & Lohr present data on what happened when the active form of folate was supplemented following the identified genetic issue with the MTHFR gene potentially affecting typical production of L-methlyfolate.

Where next with this work? Well, it stands to reason that in these days of personalised medicine percolating through to autism research and practice (see here), knowledge about a potential genetic issue identified in [some] cases of autism should be further investigated. We have other examples (see here). I'd like to see larger and more controlled trials of L-methlyfolate supplementation in relation to autism for example, based on screening for issues with the MTHFR gene. I'd like to see a few more biological measures incorporated in such study looking at other aspects of the folate and related cycles too (see here). I'd also like to see more discussion about any long-term implications and/or adverse effects associated with such supplementation along the lines of: should we really be tinkering with mechanisms linked to DNA methylation? Also in relation to some of the other diagnoses associated with issues with MTHFR there is similarly important work emerging [2] which could be quite important in certain instances...

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[1] Siscoe KS. & Lohr WD. L-Methylfolate supplementation in a child with autism and methyltetrahydrofolate reductase, enzyme gene C677TT allele. Psychiatr Genet. 2017 Mar 7.

[2] Roffman JL. et al. Biochemical, physiological and clinical effects of l-methylfolate in schizophrenia: a randomized controlled trial. Mol Psychiatr. 2017. Mar 14.

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Siscoe, K., & Lohr, W. (2017). L-Methylfolate supplementation in a child with autism and methyltetrahydrofolate reductase, enzyme gene C677TT allele Psychiatric Genetics DOI: 10.1097/YPG.0000000000000170

Acetylation focus over methylation in autism epigenetics?

The paper by Wenjie Sun and colleagues [1] (open-access) provides the blogging fodder for today's post and although based on the science of epigenetics, the usual suspect - DNA methylation - gives way to another concept: histone acetylation with autism in mind. Before heading into the paper myself, I'll draw your attention to some other write-ups of the study including a hat-tip to Jeff Craig and his piece on the topic (see here).

So histone acetylation... I've covered the subject before on this blog (see here) but basically DNA, the stuff that carries the genetic blueprint, complexes with histones to form something called nucleosomes. It's a combination likened to thread wrapped around a spool. Continuing that thread wrapped around a spool analogy, protruding threads called histone tails can be modified in a chemical sense (via processes such as acetylation where an acetyl group is added or deacetylation where one is removed) which can subsequently affect genetic transcription.

Still with me? Good. Sun and colleagues set out to look at histone acetylation in the context of autism; specifically in post-mortem brain samples donated from those deceased who were diagnosed with autism and whether they might show some important changes based on the use of "a histone acetylome-wide association study (HAWAS)."

Specific areas of the brain were assessed using the HAWAS approach - "prefrontal cortex (PFC), temporal cortex (TC), and cerebellum (CB)" - and researchers were looking for a specific type of acetylation mark called H3K27ac linked to gene activation. Based on brain samples from 94 participants ("45 ASD [autism spectrum disorder], 49 control"), a few details emerged:

• Despite the expected heterogeneity across the presentation of autism in terms of whether the diagnosis of autism was syndromic (secondary to an existing condition) or non-syndromic (idiopathic), the authors reported that approaching 70% of the autism cases "shared a common acetylome signature at >5,000 cis-regulatory elements in prefrontal and temporal cortex." In other words, a not uncommon molecular signature in relation to histone acetylation seemed to be present in quite a few of the participant samples included for study.
• Although one needs to be a little cautious about making grand, sweeping claims about how such an 'acetylome signature' comes about, the authors reported "that ASD-specific differential acetylation is driven mostly by.. factors such as environmental influences, SNPs in trans (at a different locus), indels, and larger chromosomal variants." Note the term 'environmental influences' (something I'll come back to shortly).
• When it came to what types of genes were potentially being 'affected' by acetylation, the authors report on quite a diverse spread "involved in synaptic transmission, ion transport, epilepsy, behavioral abnormality, chemokinesis, histone deacetylation, and immunity." Epilepsy and autism is a recurrent theme in the research and clinical literature (see here for example) so there are no great surprise there. 'Immunity' and autism is something else that keeps cropping time and time and time again (see here).
• I appreciate that the authors also acknowledge that whilst autism was the focus on the current work, they do also mention: "By correlating histone acetylation with genotype, we discovered >2,000 histone acetylation quantitative trait loci (haQTLs) in human brain regions, including four candidate causal variants for psychiatric diseases." This opens up the idea that various different psychiatric/behavioural labels might show 'overlap' when it comes to the histone acetylome too.

Interesting stuff by all accounts. I do like the idea that autism research is continuing to look at other areas of gene expression outside of just structural issues to the genome being linked to the condition (or should that be plural). Aside from the fact that people don't walk around with their genes permanently stuck in the 'on or off position' in every tissue all the time, the whole epigenetics field is a welcome complement to more traditional genomics. The focus on gene expression being potentially 'modifiable' might also reunite genetics and environment too (see here).

Criticisms of the Sun study? Well, brain samples from the deceased are a precious resource but not without complications when it comes their use for science (see here). I appreciate that we don't have the technology to look at histone acetylation in real-time or real-life yet with the brain in mind but one has to be cautious about the results from the brains of the deceased who may have passed away for many different reasons. There is also the temptation to move the whole epigenetics 'thing' towards acetylation on the basis of such research, but the methylome still remains potentially important (see here) and probably for more than one reason (see here). Perhaps soon we'll see a study looking at more than one epigenetic factor with autism in mind?

Going back to the concept of 'environmental influences' mentioned in the Sun paper, there are some potentially important repercussions from study results such as these. As with the concept of DNA methylation, one of the important concepts linked to the science of epigenetics is that such chemical alterations affecting the expression of DNA are potentially modifiable. This could mean that particular environmental factors working at critical periods might affect acetylation and methylation patterns and onward the expression of certain genes pertinent to the presentation of something like autism or at least facets of autism. The other scenario is that certain 'conditions' or 'interventions' might 'reverse such changes. On that last point, I might bring in some previous discussions on this blog in relation to something called HDAC (histone deacetylase) inhibitors (see here) that, as their name suggests, have the ability to inhibit the action of histone deacetylases (they remove acetyl groups). Various classes of medicines are classed at HDAC inhibitors including something called valproic acid which has some autism research history (see here for example). It's not therefore beyond the realms of possibility that the actions of certain medicines or other non-genetic factors with an influence on acetylation could be a source for further research in this area.

Independent replication is the next stage in the research process here. Alongside marrying acetylation trends with methylation trends, I do also wonder whether more functional analysis of other tissue(s) outside of just the brain might also be revealing too. I might add that traditional structural genomic issues (all those SNPs and CNVs that are talked about) can still play a role and indeed, might show some association with epigenetic issues too (see here).

And with all this talk of epigenetics and the like, due credit needs to be given to those who've been talking about this for quite a while in the peer-reviewed domain [2]...

To close, we have one final look at Rogue One before touchdown...

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[1] Sun W. et al. Histone Acetylome-wide Association Study of Autism Spectrum Disorder. Cell. 2016. Nov 17.

[2] Lasalle JM. Autism genes keep turning up chromatin. OA Autism. 2013 Jun 19;1(2):14.

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Sun, W., Poschmann, J., Cruz-Herrera del Rosario, R., Parikshak, N., Hajan, H., Kumar, V., Ramasamy, R., Belgard, T., Elanggovan, B., Wong, C., Mill, J., Geschwind, D., & Prabhakar, S. (2016). Histone Acetylome-wide Association Study of Autism Spectrum Disorder Cell, 167 (5), 1385-2147483647 DOI: 10.1016/j.cell.2016.10.031

Talking therapies impacting on the epigenetics of panic disorder?

The psychologist Oliver James has made some waves recently, coinciding with the publication of his new book, with the suggestion that nurture might be 'outdoing' nature when it comes to various concepts from intelligence to mental health. At times the recent 'debates' in this area have not been pretty as arguments about 'what the science actually says' with regards to [structural] genetics vs. environment have tended to get a little heated, and the word 'blame' being banded around quite a lot. This set in the context of views and opinions of certain relevant disciplines.

With all that in mind the paper by Ziegler and colleagues [1] (open-access) perhaps offers an olive branch between the various camps on whether genes or environment play the more important role in relation to something like mental health. Reporting results on "MAOA [monoamine oxidase A] methylation changes during the course of exposure-based cognitive behavioral therapy (CBT) in PD [panic disorder]" the authors describe how: "In a psychotherapy-epigenetic approach, responders and non-responders to a 6-week standardized CBT as defined by the number of panic attacks showed differential dynamics of MAOA methylation during the course of treatment: response was associated with a significant increase in MAOA methylation up to the level of healthy controls, while non-response rather went along with a further decrease in MAOA methylation."

In a sort of two-stage study, Ziegler et al first compared the methylation status of the MAOA gene in a small-ish sample of female participants diagnosed with PD (n=28) compared with age- and sex-matched 'not PD' controls. Actually, it wasn't just a case of looking at the gene and saying 'yes it's methylated' or not but rather looking at those little islands on a gene where a methyl group can be added and reporting findings (for 13 of them). Authors reported that as a group, the PD participants showed MAOA methylation differences compared to controls - overall hypomethylation of the gene and "at CpGs 3, 6–9, 12 and 13." Hypomethylation normally means something akin to increased gene expression; as the authors note: "As in a functional in vitro assay decreased methylation has been shown to activate MAOA expression... MAOA hypomethylation might result in a decreased availability of monoamines in the synaptic cleft and thereby confer an increased risk for PD." Authors also reported something of an interesting association between baseline PD severity and MAOA methylation levels.

In the second part of the study: "MAOA methylation was furthermore analyzed at baseline (T0) and after a 6-week CBT (T1) in the discovery sample parallelized by a waiting time in healthy controls, as well as in an independent sample of female PD patients (N=20)." The particular type of CBT administered has been detailed in other results [2] as we are also told that: "Pharmacological treatment remained unmodified during the course of CBT" and "patients were instructed to keep smoking behavior constant during the time course of therapy." Researchers reported that: "overall [in the] patient group irrespective of responder/non-responder status, as well as in the control group, MAOA methylation did not change significantly from T0 to T1 for average methylation or at any individual CpG site." When however the CBT group were sub-categorised as 'responders' or 'non-responders' on the basis of 'the number of panic attacks at T1 compared with T0' there was something a little more interesting to see. So: "responders displayed an increase in average methylation after therapy (mean change±s.e., 3.37±2.17%), while non-responders decreased in average methylation (mean change±s.e., −2.00±1.28%; P=0.001)."

Without getting too carried away with the Ziegler results and accepting that furrowed brows still accompany talk about epigenetics, the potential implications of this study could be pretty huge. I'm not completely enthralled by the 'talking therapies' it has to be said, and the 'bigging up' of the idea that they are some sort of panacea when it comes to mental health and wellbeing as a whole despite their usefulness in certain contexts. Plurality people, plurality. I am however interested when an association is made between their use and gene expression as a function of responder status even if only one gene and one condition has so far been examined with little other genetic or biochemical factors taken into account. My stance on the whole 'genes vs environment' bit too is that it is rather too simplistic to say just 'one or the other' when it comes to complicated things like human behaviour and the vast heterogeneity that underlies it. For some people it could be more of a genetic thing [2] underlying certain characteristics of a particular condition, for others it might be more non-genetic factors. The N=1 seems to be an important point.

If indeed it does turn out that the talking therapies (among various other 'environmental' factors) can impact on gene expression, there could be a few implications. The rise and rise of something like mindfulness (minus the hype) might also find a similar effect and could perhaps be [scientifically] pitted against CBT and other similar intervention hopefuls both in terms of behavioural outcomes and methylation status. Same goes for other potential 'methylation-modifiers' such as exercise for example in light of some changing attitudes in this area (see here). There is also the prospect that with some further science to do, the types of modification to gene expression could eventually be translated into a more biological intervention. Y'know, as per other discussions about the methionine cycle as a source of those methyl groups (see here) or the various agents that can affect methylation practices. Perhaps even looking at the various medications that psychiatry already has in its arsenal as having an epigenetic effect too?

More investigation is required.

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[1] Ziegler C. et al. MAOA gene hypomethylation in panic disorder-reversibility of an epigenetic risk pattern by psychotherapy. Transl Psychiatry. 2016 Apr 5;6:e773.

[2] Okbay A. et al. Genetic variants associated with subjective well-being, depressive symptoms, and neuroticism identified through genome-wide analyses. Nature Genetics. 2016. April 18.

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Ziegler C, Richter J, Mahr M, Gajewska A, Schiele MA, Gehrmann A, Schmidt B, Lesch KP, Lang T, Helbig-Lang S, Pauli P, Kircher T, Reif A, Rief W, Vossbeck-Elsebusch AN, Arolt V, Wittchen HU, Hamm AO, Deckert J, & Domschke K (2016). MAOA gene hypomethylation in panic disorder-reversibility of an epigenetic risk pattern by psychotherapy. Translational psychiatry, 6 PMID: 27045843

Meta-meta-analysing MTHFR and autism

"In conclusion, [the] present meta-analysis strongly suggested a significant association of the MTHFR C677T polymorphism with autism."

So said the findings reported by Vandana Rai [1] as yet more discussion emerges on the possible role of issues with the methylenetetrahydrofolate reductase (MTHFR) gene in relation to at least some autism. The reason I've titled this post as a 'meta-meta-analysis' is because we've previously seen meta-analysis done on this polymorphism (SNP) in relation to autism as per other entries on this blog (see here). That and other entries will also provides readers with a little more background on what MTHFR does and why it might be so important to some autism.

This time around Rai looked at 13 studies hitting the criteria for study entry covering nearly 2000 people diagnosed on the autism spectrum compared with over 7000 asymptomatic - not autism - controls. Looking at the various genetic combinations based on zygosity, the author concluded that in both Caucasian and Asian populations, there was an association between the C677T polymorphism and autism.

Where next you might ask? Well, a few possible directions are potentially indicated. First is the idea that screening for the MTHFR C677T SNP does seem to be indicated when a diagnosis of autism is received. By saying that, I'm not suggesting that science has discovered 'a gene for autism' or anything like that (not unless you, for example, count some cases of schizophrenia within that definition of autism as per other work from Rai [2]) but alongside other genetic issues associated with some autism (see here), there is a possible target gene to look at/for. Additional screening of extended family members such as parents and siblings for this SNP might also be similarly indicated, not least because of the various other 'conditions' linked to this SNP [3] and implications for preventative treatment targeting elevated homocysteine for example.

Second, as and when issues with MTHFR are detected, there are some potentially important implications for things like folic acid metabolism (yes, that has been linked to some autism too with some caveats) and potentially onwards the availability of things like methyl groups for important processes such as DNA methylation (again, something looked at with autism in mind). I might also mention some of the literature on homocysteine specifically related to autism might also be something linked here (see here) given the positioning of this compound in relation to the folate and methionine metabolic cycles. Although still in its infancy, talk of MTHFR issues when identified as being part of a 'personalised medicine' approach to autism (see here) provide an important thinking/talking point.

I'd like to think that the MTHFR C677T SNP might offer some quite important clues to at least some types of autism as and when further research is undertaken. With the continuing advances being made using the CRISPR-Cas9 gene editing tool and the ever-increasing CRISPR zoo, the modelling of the MTHFR C677T SNP is set to become quite a bit easier in times to come, and may eventually provide some important advances pertinent to autism and beyond.

Yet again, screening is the first port of call as and when a diagnosis of autism is [eventually] received...

To close, following the very sad news that Ronnie is now with Ronnie, I think it's appropriate to light four candles...

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[1] Rai V. Association of methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism with autism: evidence of genetic susceptibility. Metab Brain Dis. 2016 Mar 8.

[2] Yadav U. et al. Role of MTHFR C677T gene polymorphism in the susceptibility of schizophrenia: An updated meta-analysis. Asian J Psychiatr. 2016 Apr;20:41-51.

[3] Rajagopalan P. et al. Common folate gene variant, MTHFR C677T, is associated with brain structure in two independent cohorts of people with mild cognitive impairment. NeuroImage : Clinical. 2012;1(1):179-187.

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Rai, V. (2016). Association of methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism with autism: evidence of genetic susceptibility Metabolic Brain Disease DOI: 10.1007/s11011-016-9815-0

Methyl B12 for autism? Placebo-controlled results say maybe...

"Methyl B12 treatment improved clinician-rated symptoms of ASD [autism spectrum disorder] that were correlated with improvements in measures of methionine metabolism and cellular methylation capacity."

Those were the very encouraging results published by Robert Hendren and colleagues [1] who can now update their ClinicalTrials.gov study entry (see here). Building on the ideas that: "Children with autism spectrum disorder (ASD) have been reported to have reduced ability to methylate DNA and elevated markers of oxidative stress" (topics that have been covered on this blog before), researchers undertook a gold-standard trial - randomised, placebo-controlled - to ascertain the effect (if any) of "8 weeks of treatment with methyl B12 (75 μg/kg) or saline placebo every 3 days in a subcutaneous injection." The success of the treatment was measured by "the Clinical Global Impressions-Improvement (CGI-I) score" accompanied by "changes in the Aberrant Behavior Checklist (ABC) and the Social Responsiveness Scale (SRS)" scores. At the same time, researchers also looked at various biochemical parameters pertinent to "methionine methylation and antioxidant glutathione metabolism."

Based on the 50 children ("mean age 5.3 years") who completed the study, researchers reported a trend of improvement in autistic and related behaviours following the methyl B12 injections. Importantly, the primary outcome measure - the CGI-I scores - rated by clinicians, showed a trend of being "statistically significantly better (lower) in the methyl B12 group (2.4) than in the placebo group (3.1) (0.7 greater improvement in the methyl B12 group, 95% CI 1.2-0.2, p = 0.005)." Biological parameters also showed changes: "increases in plasma methionine (p = 0.05), decreases in S-adenosyl-l-homocysteine (SAH) (p = 0.007) and improvements in the ratio of S-adenosylmethionine (SAM) to SAH (p = 0.007), indicating an improvement in cellular methylation capacity" following the use of methyl B12 compared with placebo.

Accepting that 'subcutaneous injection' of methyl B12 is hardly a 'user-friendly' option and may very well scupper plans to use this particular intervention option for quite a few, these are potentially important results. I'm really quite interested in how vitamin B12 'vitamers' might show some links to at least some 'types' of autism (see here) including the measurement of 'brain levels' of the stuff (see here). The Hendren results suggest that quite a few more research resources might be needed in this area. I wonder also if this future research agenda would include the 'baby and bathwater' compound that is methylmalonic acid in relation to autism too (see here)?

I do also have to point out that previous research from members of this research team has not been so complimentary about the use of methyl B12 in cases of autism [2] despite the idea that there may be 'responders' to this type of intervention. To quote: "methyl B12 may alleviate symptoms of autism in a subgroup of children, possibly by reducing oxidative stress. An increase in glutathione redox status (GSH/GSSG) may provide a biomarker for treatment response to methyl B12." Such differences in reported results are not unfamiliar to autism research (the rule rather than the exception) but perhaps provides a further focus for clarification of effect.

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[1] Hendren RL. et al. Randomized, Placebo-Controlled Trial of Methyl B12 for Children with Autism. J Child Adolesc Psychopharmacol. 2016 Feb 18.

[2] Bertoglio K. et al. Pilot study of the effect of methyl B12 treatment on behavioral and biomarker measures in children with autism. J Altern Complement Med. 2010 May;16(5):555-60.

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Hendren RL, James SJ, Widjaja F, Lawton B, Rosenblatt A, & Bent S (2016). Randomized, Placebo-Controlled Trial of Methyl B12 for Children with Autism. Journal of child and adolescent psychopharmacology PMID: 26889605

Decreased brain levels of vitamin B12 in autism

I have to thank Dr Malav Trivedi for bringing my attention to some recent findings reported by Yiting Zhang and colleagues (including Malav) [1] (open-access) suggesting that: "levels of vitamin B12, especially its MeCbl [methylcobalamin] form, decrease with age in frontal cortex of control human subjects."

Further, researchers reported: "abnormally lower total Cbl [cobalamin] and MeCbl levels in subjects with autism and schizophrenia, as compared to age-matched controls." Some media on the findings can also be read here.

Working from the lab of Dr Richard Deth (quite a familiar name to this blog), researchers initially analysed a most precious sample medium (postmortem brain samples) obtained from various biobanks and including various patient groups. So alongside samples from 12 children with autism were samples from 9 people diagnosed with schizophrenia and some 43 'controls' with ages ranging between 19 weeks old and 80 years old. "Changes in Cbl species were compared with the status of methylation and antioxidant pathway metabolites" accompanied by data derived from a knock-out mouse model: "the influence of decreased GSH [glutathione] production on brain Cbl levels was evaluated in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice in which GSH synthesis was impaired, leading to a brain GSH level decrease of 60–70%."

Looking at postmortem frontal cortex brain samples, researchers reported that finding on levels of vitamin B12 - particularly the MeCbl vitamer -  decreasing with age. Bearing in mind the relatively small participant numbers included, the idea that lower brain tissue levels of total cobalamin and methylcobalamin were also present (almost unanimously) in the autism and schizophrenia groups could be important. I might at this point direct readers to previous discussions on vitamin B12 and autism on this blog (see here) including the research idea of supplementing (see here) with no medical advice given or intended.

There are a few other details worth pointing out from the Zhang findings. Analysis of thiols in brain samples across the autism vs control group revealed some potentially interesting data. So, methionine levels were quite a bit lower in the autism group [significantly lower] as were levels of "the methyl donor S-adenosylmethionine (SAM)." Both these compounds form an important part of the whole 'methylation of DNA' process (see here) among other things.

Glutathione, a compound that has seen its fair share of speculation with autism in mind (see here), was also on the research menu in the Zhang study. Interestingly and again bearing mind the small participant numbers, brain levels of this stuff were lower in the autism group as a whole but not significantly so when compared to controls. This finding might map on to other brain studies with autism in mind (see here). Likewise, cysteine (another potentially relevant compound to some autism) produced a similar finding.

I would encourage readers to take some time looking at the Zhang paper. In conjunction with other results reporting on some important elements to the emerging story (see here) I believe there are further studies to be done applicable to the notion that: "impaired methylation may be a critical pathological component" for at least some autism (see here). Indeed, other research papers have also discussed this issue [2]. The idea that studies about human ageing may likewise be informative to autism (and schizophrenia) research also carries quite a lot of traction too.

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[1] Zhang Y. et al. Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PLoS One. 2016 Jan 22;11(1):e0146797.

[2] Keil KP. & Lein PJ. DNA methylation: a mechanism linking environmental chemical exposures to risk of autism spectrum disorders? Environmental Epigenetics. 2016; 1-15.

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Zhang Y, Hodgson NW, Trivedi MS, Abdolmaleky HM, Fournier M, Cuenod M, Do KQ, & Deth RC (2016). Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PloS one, 11 (1) PMID: 26799654

HERVs as a mechanism of genetic deletion formation: relevance to some autism?

My stark lack of knowledge in the area of genetics and specifically that linked to the human endogenous retroviruses (HERVs) that litter the genome is likely to shine through in this post so be ready with that pinch of salt.

The starting point for today's post is the paper by Ines Quintela and colleagues [1] detailing a case report of "a 9-year-old female patient with autistic disorder, total absence of language, intellectual disability, anxiety disorder and disruptive, and compulsive eating behaviors." Following some genetic analysis of this young girl researchers reported on "the identification of a de novo recurrent 3q13.2-q13.31 deletion encompassing 25 genes." This in itself is interesting and adds to a growing tide of research suggesting that there may be lots of different genetic influences acting in different cases of autism; all pertinent to a more plural view of the label: the autisms.

One sentence however took my specific interest in this paper insofar as: "a 3.4 Mb recurrently altered region at 3q13.2-q13.31 has been recently described and non-allelic homologous recombination (NAHR) mediated by flanking human endogenous retrovirus (HERV-H) elements has been suggested as the mechanism of deletion formation."

As I indicated at the start of this post, the finer details of genetics are not really my forte so be warned. I was however really interested in the suggestion that the process of NAHR - when "highly similar portions of the genome wrongly recombine, deleting and sometimes duplicating a portion of the genome that lies between them" - might be linked to the presence of all/some of those fossil viruses that make us who we are [2].

The long-and-short of it is the idea that some of those little variants that we ALL have in our genome might not be all due to just chance if described as de novo (as in not inherited from mum or dad). Take CNVs (copy number variants), small alterations to the genome characterised by gains and losses in segments of DNA (I think!), as the starting point. The idea is that the location of said CNVs in relation to HERVs and other transposable elements means that there may be some kind of relationship between the two. Preliminary research has suggested that HERVs flanking particular parts of the genome might be involved in the formation of CNVs [3]. Other research has noted this process as potentially being relevant in other case reports [4] similar to that described by Quintela and colleagues where behaviour and autism have been mentioned.

Just to make the whole process even more complicated (and interesting) is the idea that CNVs when talked about with autism in mind might tend to be concentrated in 'hypomethylated' regions of the genome [5]. This brings in the potentially important process of DNA methylation (yes, epigenetics yet again) into proceedings, made further interesting by suggestions that hypomethylation of DNA = more genomic instability [6] and that certain HERVs might also to some degree be 'kept in check' by methylation means [7]. Add in some evidence of methylation issues associated with some autism (see here) and preliminary evidence of certain HERVs expression correlating with autism (see here) and comorbidity (see here), and there is the making of some potentially important hypotheses ripe for further testing.

But please, don't take my word for it.

Music: Purple Rain - Prince.

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[1] Quintela I. et al. Female patient with autistic disorder, intellectual disability, and co-morbid anxiety disorder: Expanding the phenotype associated with the recurrent 3q13.2-q13.31 microdeletion. Am J Med Genet A. 2015 Aug 29.

[2] Nelson PN. et al. Demystified . . . Human endogenous retroviruses. Molecular Pathology. 2003;56(1):11-18.

[3] Campbell IM. et al. Human endogenous retroviral elements promote genome instability via non-allelic homologous recombination. BMC Biol. 2014 Sep 23;12:74.

[4] Shuvarikov A. et al. Recurrent HERV-H-mediated 3q13.2-q13.31 deletions cause a syndrome of hypotonia and motor, language, and cognitive delays. Hum Mutat. 2013 Oct;34(10):1415-23.

[5] Li J. et al. Genomic hypomethylation in the human germline associates with selective structural mutability in the human genome. PLoS Genetics. 2012: 8: e1002692.

[6] Wilson AS. et al. DNA hypomethylation and human diseases. Biochimica et Biophysica Acta. 2007; 1775: 138–162.

[7] Lavie L. et al. CpG Methylation Directly Regulates Transcriptional Activity of the Human Endogenous Retrovirus Family HERV-K(HML-2). J. Virol. 2005; 79: 876-883

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Quintela I, Gomez-Guerrero L, Fernandez-Prieto M, Resches M, Barros F, & Carracedo A (2015). Female patient with autistic disorder, intellectual disability, and co-morbid anxiety disorder: Expanding the phenotype associated with the recurrent 3q13.2-q13.31 microdeletion. American journal of medical genetics. Part A PMID: 26332054

High risk for autism = shortened telomeres?

I don't want to spend too long discussing the paper by Charles Nelson and colleagues [1] suggesting that: "Families of children with ASD [autism spectrum disorder] who have an infant show shortened telomeres relative to families with no history of ASD" but it is worth blogging about.

As per a previous entry on telomeres and autism (see here), telomeres - the biological equivalent of plastic aglets on shoelace tips to prevent fraying - are starting to enter the autism [peer-reviewed] research psyche on top of their more traditional role suggested in ageing and cancer for example (see here). Indeed, telomeres and cellular ageing are getting quite a bit of press these days with psychiatry in mind [2] as per the goings-on with schizophrenia in mind [3] and psychotic symptoms [4].

The Nelson study started from the angle that: "Exposure to psychological stress is associated with accelerated telomere shortening, and a well-established body of evidence indicates that families with a child with autism spectrum disorder (ASD) experience heightened levels of psychological stress." They also make mention of the words 'oxidative stress' and 'DNA methylation' as also potentially impacting on telomere length and at the same time having some research 'form' when it comes to autism (see here and see here respectively).

With that all in mind, saliva samples were analysed for family members designated as 'high risk for ASD (HRA)' or 'low risk for ASD (LRA)' as a function of "older siblings' diagnostic status." Relative average telomere length was the chosen variable analysed by a "real-time polymerase chain reaction (PCR) telomere assay."

Results: "HRA families demonstrated significantly shorter telomere length relative to LRA families." This was noted across the board when it came to family members analysed (infants, older siblings parents) although the group data comparing fathers between the groups were not significantly different. The authors conclude that: "such "high-risk" families should be monitored for the physical and mental health consequences that are often associated with accelerated telomere shortening."

This is interesting work (isn't is always?) but I'm going to advise a little caution before anyone goes assuming that telomere length is the be-all-and-end-all of autism research. The inevitable hype that has followed telomere research down the years has done some real damage to the credibility of some of the findings on telomeres in other areas so one treads a little carefully. That telomere length seems also to correlate with quite a few other interesting concepts such as inflammation for example [5] is also of potential interest, particularly when inflammation seems to crop up time and time again with [some] autism in mind (see here). I dare say that future studies of telomere length and autism might want to take quite a wide view of any association including the analysis of telomerase too.

Music: The Charlatans - The Only One I Know.

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[1] Nelson CA. et al. Shortened Telomeres in Families With a Propensity to Autism. J Am Acad Child Adolesc Psychiatry. 2015 Jul;54(7):588-94.

[2] Lindqvist D. et al. Psychiatric disorders and leukocyte telomere length: Underlying mechanisms linking mental illness with cellular aging. Neurosci Biobehav Rev. 2015 May 18;55:333-364.

[3] Polho GB. et al. Leukocyte telomere length in patients with schizophrenia: A meta-analysis. Schizophr Res. 2015 Jul;165(2-3):195-200.

[4] Pawelczyk T. et al. Telomere length in blood cells is related to the chronicity, severity, and recurrence rate of schizophrenia. Neuropsychiatr Dis Treat. 2015 Jun 22;11:1493-503.

[5] Jurk D. et al. Chronic inflammation induces telomere dysfunction and accelerates ageing in mice. Nat Commun. 2014 Jun 24;2:4172.

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Nelson CA, Varcin KJ, Coman NK, DeVivo I, & Tager-Flusberg H (2015). Shortened Telomeres in Families With a Propensity to Autism. Journal of the American Academy of Child and Adolescent Psychiatry, 54 (7), 588-94 PMID: 26088664

Antipsychotic drugs as epigenetic modifiers?

The paper by Blaga Rukova and colleagues [1] (open-access available here) published last year (2014) caught my eye recently and their observations of: "major differences in methylation profiles between male schizophrenia patients in complete remission before and after treatment and healthy controls" as potential evidence that: "antipsychotic drugs may play a role in epigenetic modifications."

The process of methylation, as in DNA methylation where methyl groups are added to specific segments of the genome thus potentially altering the function of certain genes, as one facet of the emerging science of epigenetics, is something that interests me on this blog. Over and above the considerable 'hype' about epigenetics, there are some interesting details emerging from the various epigenetic studies of autism (see here for example) and schizophrenia (see here) that may well turn out to be important assuming appropriate independent scientific replication.

Rukova et al reported results based on an analysis of "20 individual Bulgarian patients with schizophrenia (8 females and 12 males) before and after treatment" whereby whole genome methylation analysis was used to look for differentially methylated regions (DMRs) pre- and post-antipsychotic use as a function of the remission or not of symptoms and gender. The results were not altogether clear insofar as some sort of methylomic biosignature linked to treatment response but researchers did report on several genes that may merit further investigation: "several new genes could be potential targets for new drug discovery: C16orf70, CST3, DDRGK1, FA2H, FLJ30058, MFSD2B, RFX4, UBE2J1 and ZNF311."

"The results from our investigation were shown statistically significant only for the male patient group in complete remission. This points out that epigenetic modification by DNA methylation is more important for male patients compared to females." This is another important detail described by Rukova bearing in mind the small participant group included for study. I'm not an expert on the role of gender/sex on response to antipsychotics but a quick look through the available research literature suggests that gender may be one of several 'non-modifiable' variables linked to therapeutic response [2] when it comes to schizophrenia. The idea that [some] males may benefit more from the epigenetic modifications potentially made by certain antipsychotics than [some] females potentially opens up a whole new world of drug discovery possibilities.

It's not necessarily new news that medicines such as antipsychotics may have quite a few more pharmacological actions than those listed on the PIL. Think about the idea that certain antipsychotics may have anti-parasitic qualities [3] as one example in light of all that schizophrenia - Toxoplasma gondii research (see here). That epigenetic properties should be added to the list of potential effects from antipsychotics [4] is also not necessarily new news.

What is perhaps important about the Rukova results outside of their focus on schizophrenia and antipsychotics is the way they looked at how epigenetics / methylation patterns might be implicated in schizophrenia and what it might mean for future research. If one were, for example, to apply a similar method of whole genome methylation analysis to say autism, and how said methylation patterns might change as a function of the myriad of interventions put forward for the condition, one could see how autism research might also benefit from such an approach...

Music: Magnetic Fields - The Book Of Love.

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[1] Rukova B. et al. Whole genome methylation analyses of schizophrenia patients before and after treatment. Biotechnol Biotechnol Equip. 2014 May 4;28(3):518-524.

[2] Carbon M. & Correll CU. Clinical predictors of therapeutic response to antipsychotics in schizophrenia. Dialogues Clin Neurosci. 2014 Dec;16(4):505-24.

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Rukova B, Staneva R, Hadjidekova S, Stamenov G, Milanova V, & Toncheva D (2014). Whole genome methylation analyses of schizophrenia patients before and after treatment. Biotechnology, biotechnological equipment, 28 (3), 518-524 PMID: 26019538

Paternal sperm epigenetic differences and offspring autism risk

"These data suggest that epigenetic differences in paternal sperm may contribute to autism risk in offspring."

So said the preliminary study results published by Jason Feinberg and colleagues [1]  (open-access) looking at "paternal semen biosamples obtained from an autism spectrum disorder (ASD) enriched-risk pregnancy cohort, the Early Autism Risk Longitudinal Investigation (EARLI) cohort." Researchers analysed 44 semen samples to ascertain whether DNA methylation differences - one type of epigenetic mechanism - might be linked to "prospective ASD development" in offspring. Said potential development of autism in offspring was measured via scores at 12 months on the Autism Observation Scale for Infants (AOSI), a schedule designed to "detect and monitor early signs of autism as they emerge in high-risk infants."

Based on some nifty technology looking at "genome-wide DNA methylation (DNAm)" Feinberg et al reported various differentially methylated regions (DMRs) in semen samples associated with offspring infant scores on the AOSI. In all, 193 sites were located where methylation was upregulated or downregulated, some of them clustering "near genes involved in developmental processes." Just in case you're still a bit baffled by all this talk of DNA methylation and what it means, I might refer you to some chatter in another area of medicine [2] and how for example, hypermethylation of a genetic site normally means gene silencing. DNA methylation might also have important implications for genetic stability too [3].

Further to their methylomic analysis of semen samples, researchers also "examined associated regions in an independent sample of post-mortem human brain ASD and control samples" to complement their study. They reported "consistent differences in the cerebellums of autistic individuals compared with controls" as a function of probes covering those "AOSI-associated DMRs" previously discussed. In summary, epigenetic differences in paternal semen DNA might transmit to offspring at-risk for autism, and that epigenetic profile may also tie into epigenetic differences found in specific brain sites of some of those diagnosed with autism. I might add that we had seen some hint that this work was coming as per some interesting data from this group presented at IMFAR 2014 (see here).

Epigenetics is an emerging area when it comes to autism research (see here). I've tried to cover quite a bit of the research with autism in mind including other studies of the methylome (see here) and some of that previous research on brain epigenetic differences potentially being linked to autism (see here). It is a complicated area and easily over-hyped, but what is perhaps so attractive about epigenetics tied into autism (and in many other conditions/labels) is the focus on gene function over and above just structural changes to the genome (and their effects) as per more traditional genetics looking solely at mutations such as SNPs. The idea is that those chemical changes to the genome described by epigenetics, affecting gene function, might bridge the gap between genetics and environment that has plagued autism research down the years (see here). That such epigenetic changes may be amenable to 'alteration' is also an interesting prospect for many people.

The Feinberg results are pretty exciting and further open up many areas of epigenetic research to autism including whether factors such as age or environmental exposure(s) might affect paternal methylation patterns (see here and see here respectively) and onwards offspring autism risk. I have to caution though that the current results were based on quite a small number of participants and covered only one part of the epigenomic landscape. Those epigenetic changes noted by Feinberg et al also need to be replicated before anyone gets ahead of themselves with the findings, bearing in mind the heterogeneity of the autism spectrum and all that elevated risk of various comorbidity (see here) potentially also coming into play.

Music: Rags To Riches - Tony Bennett.

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[1] Feinberg JI. et al. Paternal sperm DNA methylation associated with early signs of autism risk in an autism-enriched cohort. Int. J. Epidemiol. 2015. 14 April.

[2] Baylin SB. DNA methylation and gene silencing in cancer. Nature Clinical Practice Oncology. 2005; 2: S4-S11.

[3] Li J. et al. Genomic Hypomethylation in the Human Germline Associates with Selective Structural Mutability in the Human Genome. PLoS Genet. 2012; 8: e1002692.

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Jason I Feinberg, Kelly M Bakulski, Andrew E Jaffe, Rakel Tryggvadottir, Shannon C Brown, Lynn R Goldman, Lisa A Croen, Irva Hertz-Picciotto, Craig J Newschaffer, M Daniele Fallin, & Andrew P Feinberg (2015). Paternal sperm DNA methylation associated with early signs of autism risk in an autism-enriched cohort International Journal of Epidemiology : 10.1093/ije/dyv028