A cautionary tale, and a case for evaluating autism genes
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Clear targets: SFARI Gene highlights genes linked to autism and will rank their relevance to the disorder.
Imagine the following scenario: A physician sees a boy with delayed speech and diagnoses him with autism. Gold-standard clinical assessments — the Autism Diagnostic Observation Schedule and the Autism Diagnostic Interview-Revised — back up this diagnosis. An electroencephalography shows brain changes that suggest childhood epilepsy, although the boy has never had a seizure. Genetic testing of the chromosomes reveals no suspect duplications or deletions.
But, sequencing of SHANK3 — a gene that has been linked to autism — performed in a commercial laboratory, identifies a mutation in this gene: an insertion of a base pair in the eleventh coding exon, a region believed to code for protein. The mutation is predicted to lead to a frameshift — a shift of the DNA code that can create a premature stop when translated into protein.
The lab tells the boy's parents that it has identified a "predicted disease-associated mutation." Given the published evidence implicating SHANK3 in neurodevelopmental disorders, the prediction seems reasonable.
First, do no harm
The story is true, but the prediction almost certainly is not. The facts are laid out in a new paper in Brain Research from Joseph Buxbaum and colleagues and demonstrate that we need to use care when translating research findings into clinical practices.
Buxbaum and his colleagues found the same insertion in the boy's unaffected mother when they sequenced SHANK3 in both parents. This is surprising, as SHANK3 mutations tend to be highly penetrant — they almost always lead to a phenotype.
Additional data in the paper shows why. It turns out that exon 11 probably does not code for protein. It is present in the standard human reference sequence RefSeq database. However, there are no matches to it in the expressed sequence tags database, which contains DNA copies of RNA messages for the genes that are expressed. It is also absent from the RefSeq databases for mice and rats.
The authors conclude that the human RefSeq database is in error. It is certainly not the only error, given the complexity of the human genome, but it highlights the danger in using research tools to inform decision-making in the doctor's office.
This is not a new problem: similar difficulties exist in interpreting the results of genetic tests for other genes, such as BRCA1 for breast and ovarian cancer, and CFTR for cystic fibrosis.
The Brain Research paper includes a 'position statement,' which recommends that researchers establish a "large-scale collated database of genetic variation in individuals with and without neurodevelopmental disabilities," and that they should provide guidelines that can be used by clinicians, genetic testing sites and families. This statement emerged from a conference held in Toronto in September on the translation of genetic discoveries into diagnostics.
Some databases on human genetic variation already exist, including dbSNP, dbVAR, the Human Gene Mutation Database, and the ever-growing 1000 Genomes Project. These databases are not yet adequate resources for clinical use, however. The international Human Variome Project may go a long way toward addressing many problems with such databases, as its mission is to make all genetic variation related to human health and disease accessible and as correct, transparent and comprehensive as possible.
Grappling with complexity:
At SFARI, we are faced with similar complexities when interpreting the genetic results generated by surveys of the Simons Simplex Collection — a genetic database of families that have only one child with autism. Most of the data generated are of uncertain clinical relevance, and we have set up committees of clinical geneticists to determine whether an individual's personal physician should be notified of a particular finding. But, given the state of the science, these experts are simply making an educated judgment, not reaching a firm conclusion.
Although we are working to ensure that genetic databases at SFARI are error-free, difficulties in interpretation extend not only to the clinic, but to basic researchers as well. For example, the evolving SFARI Gene database, which was developed by Sharmila Basu and her colleagues at Mindspec, Inc., lists information for any gene implicated in autism in the literature. Basu and colleagues make no judgments about the strength of the evidence implicating a particular gene: they take all comers. This unbiased and comprehensive approach has a lot of merit as a first step, as it tells a user everything that has been reported.
But this can't be the last step. There is always the danger that users who are new to the field of autism genetics will view the list as a set of genes with a confirmed role in autism susceptibility. Even for more knowledgeable users, the most useful resource would be a site that critically evaluates the evidence.
With this in mind, early in 2010 we recruited a group of early-career investigators to help us develop criteria to evaluate the strength of each candidate gene and present this information to the broader community. The first of these evaluations is expected to be available online in the first half of 2011.
In addition to Basu, our advisors include Brett Abrahams, Dan Arking, Dan Campbell, Heather Mefford, Eric Morrow and Lauren Weiss. Each of these young researchers has already made major contributions to the field.
We have learned many things, but the most telling — albeit not the most surprising — is that this process is exceedingly difficult. One obvious reason is that idiopathic autism is not a single-gene Mendelian disorder in which mutations segregate perfectly in families. As for all complex disorders, we need to consider the statistics and probability of risk for each allele.
A few of these statistical arguments lead to clear results, but for most the lines are fuzzy. For example, consider the case of a common gene variant. If it is overrepresented in people with autism compared with controls to a degree that is statistically significant — reaching a p-value of 10-8 — and this result is confirmed in another data set, then the conclusion is clear.
But what if a common variant is associated at a lesser degree of statistical strength, and is still independently replicated? Or, alternatively, what if the independent replication is in a different variant of the gene? What if it is not independently replicated, but other investigators have reported rare variants in that gene in individuals with autism? What if those rare variants look like they might be functional, but researchers have not sequenced enough control genomes to be sure that the variant actually affects the gene? What if there is evidence from postmortem brain tissue from an individual who had autism that this gene's expression is altered?
How do you sensibly combine these lines of evidence? Does weak evidence in one area plus weak evidence in another area equal stronger evidence?
I could go on listing these permutations — and we have — but I trust the point is clear: there is no straightforward answer.
Guiding principles:
Given the elusive nature of the truth in evaluating genetically heterogeneous disorders such as autism, we have decided on a few basic guiding principles. First, we are putting the primary emphasis on evidence from human genetic studies, rather than functional ones. Many genomic variants may affect function, even to the point of causing seemingly relevant phenotypes in model organisms, but only a small number of these actually increase risk of the disorder in a human being.
Second, we plan to look at the evidence for each gene without prior assumptions about its strength. Sometimes the field believes that a particular gene is a risk factor, even though the evidence for this is surprisingly thin.
Finally, all of these criteria will be presented on the SFARI Gene website in a transparent manner — probably as a simple checklist. A summary of the relevant findings from the literature will appear alongside. We will then invite feedback from the community, and if the wisdom of the crowd identifies relevant literature that we have missed, or has a better interpretation of the data, the score for that gene will change. In this way we hope to arrive at a consensus that is based on the contributions of a larger group of interested researchers.
An additional outcome for this project is that it will suggest which experiments are needed to solidify the evidence for autism risk genes. Larger sample sizes, more comprehensive screening of controls and meta-analyses would all help boost confidence in the genes for which the evidence is currently quite weak — genes that are in the majority on the SFARI Gene list.
Our plan is to roll out the scores and annotations in 2011, and we invite your comments at that time.
It is important to note that this is not just an academic exercise. The case of the SHANK3 mutation mentioned above makes it clear that clinically relevant advice depends on the soundness of each link in the chain. Diagnosis and potential intervention will require not just that the identified mutation is valid, but that there is sufficient evidence that the gene harboring the mutation is actually involved in the disorder.
This is the the challenge ahead, not just for autism, but ultimately for all complex disorders in which genetic variation affects who is susceptible and who is not.
Comments
I am a Developmental-Behavioral Pediatrician in Colorado. I have recently seen a patient with a new result of a microarray study of the 16p11.2 deletion. He has speech delay, generalized hypotonia affecting writing production, endurance, and speech articulation. He also has expressive speech delay and mild social skill deficits. His parents are hopeful that this specific genetic finding will lead to more specific treatment options. We are walking that fine line between emphasizing the basics of OT, PT, Speech therapy and appropriate school accommodations while eagerly awaiting findings that a certain supplement or medication will be demonstrated as especially effective in helping individuals with this chromosomal deletion.
I want to be "in the loop" for any new information especially regarding new treatment options for the 16p11.2 entity.
The Simons Foundation is supporting an online community for families with a 16p11.2 deletion or duplication called Simons VIP Connect (http://www.simonsvipconnect.org/). The site provides families with newsletters, webinars, networking tools and other resources to understand 16p11.2 deletions and duplications.
Additionally, the Simons Foundation supports a new research initiative aimed to understand the medical, learning, and behavioral features of individuals with 16p11.2 deletions and duplications and the needs of their families. The project has assembled a team of experts at several university medical centers to collect detailed clinical information and blood samples from over 200 families. This information will help clinicians and families understand the relationship between specific genetic changes and the brain’s development. Information from the project will be stripped of any personal identifying information and made available to other qualified scientists around the world. The goal is to improve clinical care and treatment for individuals with 16p11.2 deletions and duplications as well as those with autism and other developmental disorders.
More information about the study is available on the Simons VIP Connect website under the 'Research Opportunity' tab, or on the SFARI.org site (https://sfari.org/simons-vip) or by contacting the study coordinator directly: coordinator@simonsvipconnect.org
This commentary leaves out a key element of risk - environmental factors. For instance, a 2010 Danish study of >10,000 autism cases confirmed and extended prior work in showing an association with first trimester maternal infection. Thus, to get a complete picture of causality, it will be necessary to consider gene-environment interactions. This is now being done for schizophrenia and should be done for autism.
I can only agree that there are likely to be environmental factors that contribute to the risk of developing autism, and that genetic risk factors will eventually have to be considered in the context of particular environments. This is a tall order of course. Given how difficult it is to be sure about the contributions of rare genetic variants in a complex disorder like autism (the focus of this particular commentary), the assessment of gene-environment interactions will likely be a long-term project. The good news is that some halting progress is being made, for schizophrenia (as noted above), and for other disorders like Crohn’s disease (http://www.ncbi.nlm.nih.gov/pubmed/20602997), and autism as well (http://www.ncbi.nlm.nih.gov/pubmed/21079609).
In many cases of studying ASD genetics the mother is a "confounding factor". There is increasing evidence that many females are not being diagnosed with ASD because they present "so well" and/or manifest their phenotype differently to males on which the diagnostic criteria are focused. Research is being done to improve diagnosis in females and this may well involved different diagnostic criteria. It is my suggestion that many mothers are "under the radar" for a diagnosis of ASD, but their male offspring show far greater effects for the same gene alterations. Not having adequate diagnostic tools for women with ASD makes studying ASD inheritance difficult.
I am the mother of the child with Shank3 gene mutation in your article. Actually we have found a second mutation on that gene and also a mutation on CNTNAP2 gene (my husband has the same mutation). I understand the precautions to be taken in the interpretation of theses variants. But I also believe that genetic is involved in my son's autism. I want to know why my son has autism and I understand that he is part of the first children who are being tested in private lab. with no deletion or duplication. It would be sad to discourage parents with children with small variants by telling them that these variants are not yet being understood and are probably not the cause of their autism, we are just at the beginning of great discoveries with these children, it is a challenge for science, but I trust the spirit of the future great researchers who will make grat discoveries on that field. My son, my husband and I are now part of a study at Stanford University who are growing our neurons in a dish ( disease in a dish technich) like with Rett Syndrome. I hope this study on live cells will shed more lights non my son's conditions and other children with small genetic variants. There must be a reason why these children have autism and it is certainly genetic. ( sorry for my english, I am french)
Well since my wife has chimed in, i guess i should too. I am the Father of the child in the study this article is about. I have to agree with my wife- While the article raises an excellent point concerning the neeed to correct and update available information, we personally do not feel any ill will toward the people involved-people acted appropriately with the information they had at the time. We have understood from the beginning that much more is needed to fully understand what is going on with our son.
We are far more concerned that this "cautionary tale" will be used as more impetus for parents to not investigate the genetic issues involving their children with autism. Irregardless of whether exon 11 codes for the protein or not, the variant seen is still very rare. The original laboratory has now told of of another variant of unknown significance in the Shank 3 Gene in another exon. While the original variant has since been found in my wife and is therfore not De Novo, as originally thought, we still do not know what the status of thie second variant is-It was not found in my wife. Sequencing was done for my Shank 3 alele, but not for the region of the second variant.
Also, the laboratory found another variant of unknown significance for CNTNAP2 in my son's as well as my sample. So far, only three Autism suspect Genes have been sequenced for my son-cdkl-5 being the last and in which no variant was found. So what are the odds of that? We need more testing to fix errors in the data bases-not less. We need more testing to start the process of looking at multiple gene associations-thought by most researchers to be causitive. Otherwise parents will continue to talk about Danish studies and the "Evidence of Harm" found during a "Silent Spring". We need to encourage parents to seek real science- not junk.
I have seen a baby boy who has a speech delay. Till 2-years, he did not speak a word. But after when he turned about 21/2 years, he began to speak and talked normally. His parents are happy. I wonder what is the cause & effect.
To Naomi Bishop: You raise a good point. John Constantino (http://www.ncbi.nlm.nih.gov/pubmed/21289537) and others have pointed out that females in families affected by autism are more than 10 times as likely to exceed the first percentile for autistic social impairment than females in the general population, and yet they very often do not get a clinical diagnosis. The differential effects of strong genetic risk factors in males vs. females remains one of the key unanswered questions in the field.
To Marie and Richard Fauth: First, thank you for writing and sharing the experiences of your family. Your perspective will no doubt be of great interest to our readers. The commentary was written in the context of a fair amount of hype in the popular press regarding the predictive power of genetic tests for disorders where our understanding is still rather limited. My concern was that we should be clear-eyed about our ability to interpret this information at such an early stage. That said, we are in complete agreement that the answer to problems with databases and uncertainty in genetic testing is more research, not less, and more participation by determined and well informed parents like you, not less. Indeed, much of the research that SFARI supports depends on the active involvement of families who have been affected by autism. It’s exciting to hear that additional sequencing has identified some new variants of interest. We share your trust that great discoveries are possible.
Thank you Alan for your kind response. Certainly we should all view new information with a degree of skepticism. However, we believe the "hype" in which you are referring to be marginal-at best. The "hype" that continues to be a huge problem for parents results in parents feeding their kids all kinds of unproven substances and going to third world countries for stem cell treatments. Certainly envoronmental factors play a role- but the effectivenmess of most, if not all, of the information out there concerning environment as well as diet has been hyped to the point where it is blatantly false. I appreciate the conservative approach of real science to do no harm. However, an overtly cautious view also leaves open a vacuum filled by outright lies. A vast resource of energy and support has been hijacked by those who do not follow the ethics or rigorous methodology of real science and real science needs resources. Its time to take it back.
Thank you for posting this article. I was glad to read Naomi Bishop’s comment concerning women and autism, I believe it could be a link concerning my son an I genetic mutation. I am asking parents who has children with autism to look into genetic for answers. It is very hard to educate people on a subject as hard as genetic, parents have to educate themselves as much as they can to try to navigate into the field of genetic and autism. I know parents who spend 30 thousand dollars on stem cells treatments in South America, I know parents who spend 50 thousand dollars within years on different types of medical treatments. There are millions of parents everyday, in the world, who are spending this amount of money to help their children. I think this money should go for genetic testing, genetic research…and Education. Most everybody can understand what a syndrome is, but not everybody understand small variants. The sad part is that it’s impossible to find doctors who understand small variants without having to reach scientists. I agree that there is a need to educate physicians and parents about autism and genetic. I looked into projects like IAN or Autism genome project, but our family didn’t fit because we only have one child, and it didn’t seem to me that you had control on your child’s testing. Our only options was to go with a private Lab and they will be more parents like us who will use private testing. It’s very expensive right now but the cost will eventually go down. They will be more parents like us who will try to seek for help in the medical and scientific world. Parents, Doctors and researchers need to be ready to communicate on specific cases who are our children. There is a strong need to help people who has children with autism, with no deletion or duplication. People who have a hard time testing their children for genetic because they can’t find a doctor who will help them to do at least a micro array analysis, they only test doctors propose is FISH and if FISH comes back normal, everybody is done after that. FISH is not enough to test children with autism, micro array analysis should be part of the process, even micro array analysis is probably not enough to test for very small variants. Our World is not stable and relativity is a strong part of how it functions. I believe that they might not be a complete answer on how small genetic variants in DNA works. Genetic Databases are important to help understanding the mapping of autism. But we need to ad other type of research to complete all the data, like disease in a dish technology or brain imaging. I am being patient concerning my son and other children with autism with no deletion or duplication. I know it’s a long and hard task for science and they are so different cases in the world. Still, I am also excited to see the growing interest concerning genetic small variants and autism. I want to thank everybody who are helping families like us to navigate the fields of genetic and science. I truly believe we are all sharing the same excitement for the future outcomes. ( Sorry again for my English, I am French)
A Cautionary Tale about a Cautionary Tale
As the parents of the child in the Buxbaum study have already explained, the Shank3 variation in this case study is back on the diagnostic radar. Those who study genetic causes of Autism have always been aware that different gene errors have different phenotype penetrance. It is not clear that "SHANK3 mutations tend to be highly penetrant". This is a misinterpretation of literature, which still has rather few studies and cases of pure SHANK3 errors. SHANK3 also has a very large number if intragene promotor regions, which means that strange variants of SHANK3 can arise with a frame shift or other error. Little is known yet about these variants. There is one variant in mice that has a dramatic pathological effect on synaptic protein regulation.
What is known is that the Buxbaum group announced this Cautionary Tale before the discovery of a second gene error. That second error is matched in the other parent, who is unaffected. Thus, the most parsimonious explanation is that the two Autism-related genes, one from the mother and one from the father, operate in concert to induce the phenotype in the child. Thus, the original interpretation, that the SHANK3 error in this child is a causal factor for Autism, remains the best.
Everyone agrees that much more work is necessary, both to clean up existing databases and develop relevant new ones. Everyone agrees that clinical inferences are always made in the light of available information. It is the responsibility of the clinician to appreciate the limits of available information. However, the emerging evidence that SHANK3, and other genes, have a causal role in Autism should not be discarded when a single case study is difficult to explain. To their credit, the Buxbaum group chose words carefully and did not conclude more than their evidence allowed.
People should read the "Cautionary Tale" elaborated in the Buxbaum paper with Caution. If you look carefully, only some of the authors signed onto the "position statement"; not all were comfortable with it. In retrospect, the conclusion of the paper, that SHANK3 is not a causal factor in this case of Autism, was hasty. The authors imagined that some other gene was causal. They overlooked what is now the best explanation. Both SHANK3 and CNTNAP2 (both causal Autism-related genes) conspire in this child's Autism. In fact, this result is in concert with the greater number of DNA variants seen in Autism in general. It is also consistent with the evolving notion that Autism is the breakdown of synaptic structural components, which may occur from a single cause, or multiple causes. (I include, here, environmental causes.)
The Buxbaum group has corrected the RefSeq database, they have studied many clinical cases in depth, and they have recently published a detailed investigation of Shank3 in a mouse model. The Cautionary Tale, is a small misstep, that has been blown out of proportion. Let us all be careful with our interpretation of the available information, including those who would caution us.
Andy Mitz
Parent of a child with a Shank3 deletion
Without a clear ethical consensus and intention for this resource, the problem of complexity can only take on a life of its own. Complex problem solving is a specialism needed here.
Whilst I see you have designed a contained system of evaluation based on democracy, perhaps you fail to take on the wider ethical issue by devolving direction back to the contributor as it were. Perhaps this responsibility should be properly public property, the wider gene pool is in a sense a national asset. This disowning mechanism grants freedom beyond what might normally pass for an individual entity. I have to be suspicious of the motive of this mechanism, sorry. The State has a special interest also perhaps undeclared.
Apology to Buxbaum and Colleagues
In re-reading my post, I realize that I may have given the impression that Dr. Buxbaum and his colleagues were either not careful or not thorough in their work, or not wise in their cautionary note. I apologize for leaving this impression. The caution they express was, and remains, valid and important. I do not question their interpretation of the available results. I did want to emphasize that the reader should approach ongoing research with caution. In this medical case, SHANK3 may still play an important role. I was concerned that we cannot decide on the role of SHANK3 until the case is more completely understood. Regardless, I apologize to Dr. Buxbaum and his team for making my post sounding so negative about their work. In fact, I hold their efforts and abilities in high esteem and appreciate their dedication to not only understanding the causes of ASDs, but also their dedication to translate research into effective treatments.
Andy Mitz
Parent of a child with Phelan-McDermid Syndrome
Mouse models of Shank3 show that if you delete the N-terminus portion (including exon 11) you will still see expression of the remainder protein because there are internal promoters in Shank3.
A more C-terminal Shank3 mutation that will delete the C-terminus of Shank3 will result in a more severe Autistic phenotype like the group at Johns Hopkins have shown.
Thank you for responding. I also can appreciate your wanting to remain anonomous. However, perhaps a nom de guer would be more appropriate as readers would know that the individual responder is the same should additional comments be left. Your information is interesting. However it is my understanding that multiple mouse models have now been developed that knock out various differant parts of this gene. In fact, with the exception of exons 10 to 13 models have now been developoed that knock out the whole gene-see duke and mit research. Your point on severity is however interesting as my son would likely not be considered severly autistic. He is very affectionate and smart. He would likely be considered high functioning if he could talk. Most consider him moderate. I also note that some of the original research implicating shank3 and autism included children with mutations in exon 11 as did a japanese study. In fact the japanese study had a mutation with an adjacent codon position to my son's frameshift-not even one base pair in between.
I met a 4 year old this weekend with a 22q13 chromosonal deletion. While the child has some anxiety issues-for the most part he is very high functioning. Interestingly, the deletion did not affect shank 3. I.E his shank 3 gene is intact.