Friday, March 23, 2007

A Tale of Two Studies – Part the Second

”…it was the epoch of belief, it was the epoch of incredulity, it was the season of Light, it was the season of Darkness, it was the spring of hope, it was the winter of despair, we had everything before us, we had nothing before us, we were all going direct to Heaven, we were all going direct the other way…”


We now return to our previous story…


Strong Association of De Novo Copy Number Mutations with Autism.

Sebat J, Lakshmi B, Malhotra D, Troge J, Lese-Martin C, Walsh T, Yamrom B, Yamrom B, Yoon S, Krasnitz A, Kendall J, Leotta A, Pai D, Zhang R, Lee YH, Hicks J, Spence SJ, Lee AT, Puura K, Lehtimaki T, Ledbetter D, Gregersen PK, Bregman J, Sutcliffe JS, Jobanputra V, Chung W, Warburton D, King MC, Skuse D, Geschwind DH, Gilliam TC, Ye K, Wigler M.

Science. 2007 Mar 15; [Epub ahead of print]


In this study, the authors used ROMA, a type of comparative genome hybridization, to look for de novo mutations in autistic children. They looked at 195 autistic children and their parents (need to look at the parents’ genome to know if the mutation was inherited or arose spontaneously) and 196 unaffected children and their parents.

What they found was – to put it mildly – very interesting.

The size of the human genome limited the resolution of the technique (in this study) such that they could detect only about 15% of the mutations that cytogenetic techniques indicate to be present. Even with these limitations, they found:

[1] Spontaneous mutations were more frequent in patients with autistic spectrum disorder (14/195) than in unaffected individuals (2/196).

[2] The frequency of spontaneous mutations was higher (12/118) in sporadic cases of autism – children with no affected relatives – than in cases from multiplex families (2/77).

[3] The frequency of spontaneous mutations in unaffected individuals was 1% (2/196).

[4] Most of the mutations in autistic individuals were deletions (12/15 – one child had two mutations), while the two mutations in the controls were duplications.

[5] None of the mutations were seen more than twice and most were seen only once.

[6] The mutations seen in autistic children were on chromosomes 2, 3, 6, 7, 10, 12, 13, 15, 16, 20 and 22.

If, as they suspect, their technique was only able to pick up 15% of mutations, then the actual mutation rate in autistic children may be much higher.

What it means is that there is a statistically significant (p less than 0.0005) association between spontaneous mutations and autism. Now, I've often argued that association is not causation, and so do the authors of this study.

However, the locations of many of the mutations were highly suggestive of a connection. For instance, one mutation - a 1.1 Mb deletion from chromosome 20 - deleted 27 genes, including the oxytocin gene OXT. This is particularly notable, as oxytocin has been shown to regulate social behavior and awareness.

Five of the mutations involved only a single gene, which indicates that these genes are prime candidates for further study.

Now, what does this mean for the people who claim that autism can’t be genetic, that it’s all the result of exposure to (fill in the blank with environmental toxin de jour)?

Well, it means that they need to start thinking up a way to “spin” these results.

Doubtless, they will find that some of the authors have – at one time in their careers – been supported by the pharmaceutical industry (“Big Pharma”) or “the government” or maybe they’ve ridden in black helicopters.

Or they’ll find some other equally ridiculous claim of bias, corruption or conspiracy.

But they won’t be able to stem the tide.

"What tide?", you ask.

There’s a tidal wave of data bearing down on the “reality deniers” in the autism world, and they’d better start heading for higher ground.

Or learning how to swim.


Prometheus


Note: Prometheus will be attending a conference of minor mythological figures next week and will not be able to moderate comments. Rest assured, when he returns, all pending comments will be dealt with in a firm but fair manner.

11 Comments:

Anonymous Mendel and Monod said...

But wait, isn't the brain as easy to understand as wrinkles on a pea plant and the control of the lac operon?

It's easy, the environmental cofactor that we must invoke is....

23 March, 2007 20:58  
Blogger ruidh said...

I appreciate your efforts to shine a light of reason autism claims, but isn't there something more to this story? Let's assume for the moment that the deletions are the proximate cause of autism. Could exposure to one or more environmental toxins cause these kinds of mutations? Considering the large amount of chemicals we find ourselves in contact with on a daily basis, I find it at least plausible that one of them has unintended and unknown effects. That's not to say that it would ever be easy or even possible to find the culprit through scientific means. But, shouldn't we at least keep an open mind to the possibility of an environmental cause?

24 March, 2007 00:20  
Blogger Prometheus said...

Ruidh,

Environmentally-induced mutations would have to have occurred when the child was a single cell, unless you are arguing that an environmental factor (such as a "toxin") was able to produce exactly the same mutation in each cell (the tests were done in replicate).

A better conclusion is that something (age, environmental exposure, random events) caused a mutation in the germ line (i.e. egg or sperm) of one of the parents.

This would not cause a change in the DNA of the parents (except for the sperm or egg that combined to form the autistic child) and so would cause a de novo mutation in the child.

So, unless you want to argue that the exposure occured in the few hours between fertilization and the first few cell divisions, environmental exposures to the child could not have caused the mutations seen.

Mendel and Monod;

I'm not sure what you're trying to say here. To be sure, the genetics of brain development are not as well understood as the wrinkles on a pea or the lac operon, but that doesn't mean that they don't follow the same rules.

For that matter, we do understand a great deal more about gene regulation and expression than either Mendel or Monod did. And, given more time and research, we may well understand quite a bit more about brain development.

Which environmental factor did you have in mind?


Prometheus

24 March, 2007 16:21  
Blogger daedalus2u said...

It is not clear to me that this study sheds light or heat on the cause(s) of autism or ASDs. Most of the ASD cases didn't have detectable changes (181/196). Their ASD must have been caused by something else.

I appreciate that the probes were not sensitive enough to detect all changes, but that also applies to the controls. If one argues that the ASD cases "must be" higher, then the controls "must be" higher too. How much higher?

Second, they looked at DNA from blood cells, not genomic DNA. Blood cells are susceptible to somatic mutations, that is what causes leukemia. There are lots of environmental exposures that will cause leukemia, solvent exposure being one. Solvent exposure causes oxidative stress via expression of cytochrome P450 enzymes. ASDs are characterized by increased oxidative stress. Leukemia may be associated with increased oxidative stress:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12969817&query_hl=30&itool=pubmed_DocSum

Leukemia requires specific deletions of oncogenes, not the generalized/random deletions they observed. Are what they seeing "subclinical" blood cell genetic instability?

Is what they observed simply an association with the "real" cause(s) of ASDs? Low nitric oxide?

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15155057&query_hl=38&itool=pubmed_docsum

Low nitric oxide does explain the neuronal hyperplasia observed in ASDs.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11698593&query_hl=41&itool=pubmed_docsum

25 March, 2007 18:10  
Blogger Maya M said...

BTW have you heard about the findings of Reichenberg et al. that children of older fathers are more likely to be autistic (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16953005&query_hl=11&itool=pubmed_docsum)? Paternal age is a known factor predisposing to gene mutations.

26 March, 2007 04:05  
Blogger ruidh said...

I think I was just confused. I was conflating developmental defects with genetically caused mutations. Your point about the potential exposure being to the parents is well taken. Thank you for the clarification.

26 March, 2007 05:43  
Blogger concerned heart said...

I would guess the CNVs are in the spermatagonia of the fathers. Autism is much more prevalent, the de novo version, in the children of older and maybe chemically exposed fathers,. I say assay the sperm and ancestral spermatagonia next.

Go to the EBD blog if you want easy links for the sources.

http://ebdblog.com/paternalage/

What do you think?


Fathers’ Age as Contributor to Risk for Autism
Leslie Feldman
The average age of fatherhood is increasing in the US and in Western Europe. Some research shows that offspring of older fathers are at increased risk for diseases and conditions (Bray et al., 2006). Some experts predict an upswing in cases of schizophrenia will accompany the increasing average paternal age. “The actual percentage of cases with paternal germ line-derived schizophrenia in a given population will depend on the demographics of paternal childbearing age, among other factors. With an upswing in paternal age, these cases would be expected to become more prevalent” (Malaspina et al., 2006). Approximately 25-33% of all cases of schizophrenia may be due to the father’s age at conception, according to Malaspina (2006). Malaspina sees a connection between advancing paternal age and neural functioning difficulties in people with autism and with schizophrenia. According to Tarin et al. (1998), there are well over 30 known conditions that the offspring of older fathers are more at risk for (see chart on paternal aging in the linked article).

The diagnosis of autism is increasing in the US and elsewhere (Centers for Disease Control, 2006). In a population study of 1990 through 1999, a total of 669,995 children, Atladóttir and colleagues (2007) reported increased diagnosese of autism, Torrette Syndrome, and hyperkinetic disorder. Is there a connection between increased cases of disorders such as autism and increased average paternal age? Psychiatrist Michael Craig Miller (2006), editor of the Harvard Mental Health Letter is convinced there is. Although a connection between the two would be corelational (not causal), the relationship encourages examination of the possibility that something related to paternal age (e.g. mutations in gametes) may contribute to the occurrence of autism. If there is a potential causal relationship, the new study by the Centers for Autism and Developmental Disabilities Research and Epidemiology (CADDRE) Network would provide a valuable opportunity to test the hypothesis.

Observations of a connection between advanced paternal age and difficulties for offspring go way back. Earlier research looking for a link between maternal age and autism also found the average paternal age (34) was much higher than the average age in the general population (Gillberg, 1980). Geneticist James F. Crow (1997) cites Wilhelm Weinberg (1862-1937) as noticing, during his 42 years of medical practice and helping 3,500 births, that the mutation rate might be a function of paternal age. Crow said, the evidence suggested that the greatest mutational health hazard in the population is fertile old men.

A study by Reichenberg et al. (2006) found a strong connection between cases of autism and advancing paternal age. Reichenberg and colleagues, who found more autism as paternal age increased, also found that the ratio of girls to boys in this cohort was 1:1, suggesting that this was a special subset of autism, maybe de novo rather than familial autism.

What might be the mechanism that produces higher rates of disorders among children of older fathers? The DNA in a 20 year-old male has been copied approximately100 times but in a 50 year-old father it has been copied over 800 times. Singh and colleagues (2003) studied differences in the sperm of older and younger men. Men over age 35 have sperm with lower motility and more highly damaged DNA in the form of double-strand breaks. The older group also had fewer apoptotic cells, an important discovery. (Apoptosis is form of cell death that protects the parent organism from problems or that permits differentiation, as in resorption of a tadpole’s tail.) A really key factor that differentiates sperm from other cells in the body is that they do not repair their DNA damage, as most other cells do. As a result, the only way to avoid passing DNA damage to a child is for the damaged cells to undergo apoptosis, a process that the study indicates declines with age. Singh is quoted in Science Blog (Sullivan, 2002) as explaining that, “In older men, the sperm are accumulating more damage, and those severely damaged sperm are not being eliminated.”

Sources

The following list of sources is for works cited in this document or for other studies finding a connection between age of fathers at conception and various disorders. Access to some of the Web-based resources may be limited because of the policies of the publishers.

Atladóttir, H. O., Parner, E. T., Schendel, D., Dalsgaard, S., Thomsen, P. H., & Thorsen, P. (2007). Time trends in reported diagnoses of childhood neuropsychiatric disorders. Arch Pediatr Adolesc Med., 161, 193-198. Link

Brown et al. (2002): Paternal age and risk of schizophrenia in adult offspring. Am J Psychiatry, 159, 1528-1533. Link

Bray, I., Gunnell, D., & Smith, G. D. (2006). Advanced paternal age: How old is too old? Journal of Epidemiology and Community Health, 60, 851-853. Link

Burd et al., (1999). Prenatal and perinatal risk factors for autism. J. Perinatal. Med., 27, 441-450. Link

Byrne, M., Agerbo, E., Ewald, H., Easton, W. W., & Mortensen, P. D. (2003). Parental age and risk of schizophrenia, A case control study. Arch Gen Psychiatry, 60, 673-678. Link

Centers for Disease Control, (2006). How common are Autism Spectrum Disorders (ASD)? Link

Centers for Disease Control. (2002). Prevalence of the Autism Spectrum Disorders (ASDs) in multiple areas of the United States, 2000 and 2002. Atlanta, GA: Author. Link

Crow, J. F. (1997). The high spontaneous mutation rate: Is it a health risk? Proc. Natl. Acad. Sci. USA, 94, 8380-8386. Link

Dalman, C., & Allebeck, D. (2002). Paternal age and schizophrenia: Further support for an association. Am J Psychiatry, 159, 1591-1592. Link

Gillberg, C. (1980). Maternal age and infantile autism. J. Autism and Developmental Disorders, 10, 293-297. Link

Lauritsen M. B., Pedersen, C. B., & Mortensen, P. B. (2005) Effect of familial risk factors and place of birth on the risk of autism: a nationwide register-based study. J. Child Psychology and Psychiatry, 46, 963-971. Link

Miller, M. C. (2006) A new key to Autism. Aetna IntelliHealth, September 25. Link

Malaspina, D., et al. (2001): Advancing paternal age and the risk of schizophrenia. Arch Gen Psychiatry, 58, 361-367. Link

Malaspina, D. (2006). In session with Dolores Malaspina, MD, MSPH: Impact of childhood trauma on psychiatric illness (interview by N. Sussman). Primary Psychiatry, 13(7), 33-36. Link

Malaspina, D. (2006). Schizophrenia risk and the paternal germ line. Schizophrenia Research Forum. Link

Rasmussen, F. (2006) Paternal age, size at birth, size in young adulthood&mdashrisk factors for schizophrenia. Eur Journal of Endocrinology, 155 Suppl 1:S65-69. Link

Reichenburg, A., Gross, R., Weiser, M. Bresnahan, M., Silverman, J. Harlap, S., et al. (2006). Advancing paternal age and autism. Arch Gen Psychiatry, 63, 1026-1032. Link

Singh, N. P., Muller, C. H., & Burger, R. E. (2003). Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertility and Sterility, 80, 1420-1430. Link

Sipos, A., Rasmussen, R., Harrison, G., Tynelius, P., Lews, G., Leon, D. A., et al. (2004). Paternal age and schizophrenia: A population based cohort study. BMJ, 329, 1070. Link

Sullivan, B. J. (2002). Research reveals a cellular basis for a male biological clock. Science Blog, 2002-11-25 22:31. Link

Tarin, J. J., Brines, J., & Cano, A. (1998). Long-term effects of delayed parenthood. Human Reproduction, 13, 2371-2376. Link

Tsuchiya, K. J., Takagai, S., Kawai, M., Matsumoto, H., Nakamura, K., Minabe, Y., et al. (2005). Advanced paternal age associated with an elevated risk for schizophrenia in offspring in a Japanese population. Schizophrenia Research, 76, 337-342. Link

Wohl, M. & Gorwood, P. (2006). Paternal ages below or above 35 are associated with a different risk for schizophrenia in offspring. Eur. Psychiatry, Dec 1 [Epub ahead of print]. Link

Zammit, S., Allebeck, P., Dalman, C., Lundgerg, I., Hemming, T., Owen, M. J., et al. (2003). Paternal age and risk for schizophrenia. Br. J. Psychiatry, 183, 405-408. Link

24 Comments »

26 March, 2007 15:06  
Blogger Prometheus said...

Daedalus2U,


Granted - 10% of simplex ASD cases had de novo mutations vs 1% of unaffected individual. While there is no a priori reason to suspect that the number of mutations found won't go up in both groups with greater resolution, it still looks like de novo mutations are significantly more frequent in ASD patients.

Secondly, I'm not sure what you mean when you say "...they looked at DNA from blood cells, not genomic DNA..." They looked at DNA from white blood cells, which would be genomic DNA, unless you have a different definition of "genomic". I'm not sure that blood cells are more susceptible to "somatic mutations" than any other cells are - they divide more often than most cells, which makes them more likely to express the effects of mutations than, say, neurons, which rarely (if ever) divide.

Prometheus.

28 March, 2007 19:16  
Blogger concerned heart said...

Prometheus-
If they now looked at the DNA in the paternal germ line, the DNA in the spermatagonia and sperm of the fathers of the children with the CNVs, they might find the copy number variations there. The sperm and spermatagonia collect spontaneous mutations that will not show up in any other the father's other cells. Even greater than three cups of coffee a day cause double strand breaks in sperm DNA aging causes single strand breaks in DNA and alkali labile sites in DNA. The DNA in sperm are very vulnerable to mutations that are subsequently passed on to offspring and future generations.The DNA of the genes controlling the development of the nervous system seem to be especially sensitve to mutations.
Try reading this article on 3 generations of rats by Maurice Auroux et al.

http://www.sciencedirect.com/science/article/B6T2C-47P87KN-7G/2/fdda2c3118ba36ab349cf9807bb44a37?&zone=raall
I hope this clarifies why I believe that Sebat and Wiggler et al. should use the
microarray technology – the high-resolution method for analyzing DNA on the father's spermatagonia and sperm.

30 March, 2007 12:35  
Blogger daedalus2u said...

Prometheus,

I was sloppy (and actually wrong) in my articulation. White blood cells do of course have genomic DNA, what I meant was that the pool of cells that form blood cells does clonally replicate many more times than other somatic cells, such as neurons. Due to Mueller's Ratchet, defects in replication will accumulate more in blood cells (where they likely have little effect on neuroanatomy or other physiology), rather than in cellular compartments known to be abnormal in ASDs. Leukemia results from DNA damage in the pool of cells that form blood. Finding DNA damage in blood doesn't prove that there is DNA damage in neurons. Of course, neural biopsies are too invasive and damaging to do these experiments on living humans.

If the paper had given the ages of the different individuals, or had shown a plot of copy number variations vs age, we might be able to appreciate if the copy number mutations occured in utero, or later. Looking at DNA from different tissue compartments might also allow understanding of when the de novo mutations occurred, and whether it was before or after the formation of neuronanatomy associated with ASDs.

I suspect that your own liver, having to regenerate itself every day has accumulated many more mutations than other parts of your anatomy.

The paternal age effect on ASDs, while I don't dispute the data, could have causes other than DNA damage. The paper reported no such effect of maternal age (yes, there is the difference in replication between oocytes and sperm percursors, however even in the absence of replication, exposure to radiation and mutagens continues). Second, presumably any paternal DNA damage would be heritable, even if "sub-clinical". Even modest DNA deletions over a few thousand generations would be quite deleterious.

A speculative explanation, is that having an older male as the father of a fetus in utero may be a source of "stress". It was not too long ago, that humans routinely practiced infanticide when the old alpha male was displaced (see the Bible, Numbers 31:17-18). Since an old alpha male is more likely to be displaced than a young alpha male, there may be a "stress" response based on paternal age, irrespective of maternal age. The study was reported out of Israel, which is a high "stress" environment, due to the various conflicts in the region.

If, as I suspect, ASDs are caused by maternal stress in utero, that could rationalize both the older father effect (subconcious maternal stress due to potential new alpha male infanticide), and also a young father effect (stress due to being a young mother and trying to hide a pregnancy). Presumably the U-shaped curve effects can't be due to accumulation of DNA damage.

http://humrep.oxfordjournals.org/cgi/content/abstract/4/7/794?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=1&author1=Auroux+M&andorexacttitle=and&andorexacttitleabs=and&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=relevance&resourcetype=HWCIT

Experiments with knock-out mice have shown that the genome is remarkably robust. "Approximately two thirds of all knockouts of individual mouse genes give rise to viable fertile mice."

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10421576&query_hl=6&itool=pubmed_docsum

I think I agree with the observation expressed by EBD blog, that the increase in DNA damage in sperm of older men may relate to decreased apoptosis of damaged precursors. That might hold for younger fathers too.

In my own mind, I make a "big deal" about monozygous twins being discordent for ASDs. To me, that shows that environmental effects are important. Dizygous twins have greater concordance than full siblings. It is extremely difficult to separate out in utero environmental effects from genetic and from epigenetic effects. No doubt they are all important.

31 March, 2007 12:16  
Blogger Prometheus said...

Your concern about the errors accumulating in the DNA of white blood cells is valid, but the technique they used would have "averaged out" the errors.

They did not use a single WBC to form a clonal group (although they did use EBV-immortalized B-cells in some subjects, they did not isolate a single cell prior to EBV treatment), which means that - unless you argue that the DNA errors are all in the same place, the resultant DNA they tested would have had a broad range of errors. However, ALL the DNA strands would have had any errors (copy number variations) that were present in the original genome.

The use of microarray hybridization to compare the parents, children and siblings would then "average out" random errors and show only those errors (variations) that were present in all (or a significant fraction) of the cells.

I make no comment on the possible reasons for the copy number variations, as they are literally too numerous to mention.

As for monozygotic twins being discordant with regard to autism, the latest information I have is that monozygotic twins are 60% concordant for autism and 90% concordant for any ASD (autism spectrum disorder), whereas dizygotic twins are less than 1% concordant for autism and 10% concordant for ASD. No matter how you look at it, autism (and ASD) have a huge genetic component.

Finally, the question of whether autism is a single disorder. All indications are that autism is not a single disorder and so would have a variety of different causes. Perhaps there is a subset of autistic children whose autism is environmental. We have yet to isolate this group or find an environmental cause. Prematurely blaming one compound (or element) out of millions seems unwise at this stage.


Prometheus

02 April, 2007 12:12  

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