Journal Club: Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations

 Hey y’all! Welcome to my first ever ever post on this new blog. I’m starting out with a journal club feature--a close reading of an interesting or recently published paper in a field I hopefully know a little about.

Today I’ll be reading and writing and working through this paper. PMID: 21572417

(**don’t know what a PMID is? Check out my frequently used acronyms page here)

First off, a little bit of background. Autism aka ASD, or Autism Spectrum Disorder, is an extremely complicated disorder, diverse in phenotype, and the genetic etiology is wildly unknown. There aren’t even really many candidate genes that people can agree on yet. We’ll touch more on that later as we get through this paper. This paper, specifically states that, “ASDs are characterized by pervasive impairment in language, communication and social reciprocity and restricted interests or stereotyped behaviors. There have been some GWAS hits for Autism, and there have been a few rare variants of large effect that have also been described, but for the most part the genetic basis for the vast majority of autism cases remains unknown. And for the stuff that is known, it’s a long way away for being useful information to the general public (read: clinical setting). And long way is an understatement. It’s a really really long way away.

Hypothesis

What O’Roak et al. hypothesizes is this: that sporadic cases of ASD, that is, families with only one child with autism with no family history, that seemingly came out of genetic-nowhere, are more likely to be as a result from a de novo, or new, mutation (not inherited from either parent), as opposed to families with multiple affected individuals, which should more likely result from inherited variants.

Now I’ma let you finish: but hold on a minute. There is a huge, huge assumption that they are making here. Which is that cases that seem sporadic, may not actually be sporadic. Families that have a first child with autism are a lot less likely to have more kids. And if they have two kids who are born with autism there are really really less likely to have more kids after that. (Like those scientific terms I used there?) So discovering cases that are truly familial are really hard. The fact that this hypothesis only focuses on the rare variant model of disease etiology is another shortcoming, but you can’t really fault this paper for that...yet. You can only test on hypothesis at a time, after all. What’s bad is when you limit yourself only to that hypothesis in the results and discussion. But we haven’t gotten there yet. </end Kanye interruption>

Methods

20 Autism trios: this is generally two parents and an affected kid. Their clinical evaluations can be found in the supplementary information. Here’s another source of bias. ASD is such a diverse disorder, thus the inclusion of the word “spectrum” in it’s name. As stringent and as uniform as the diagnostics manual tries to be, there is still a lot of wiggle room that is so dependent on the clinician. I know a clinician that pretty much diagnoses anyone who’s the least bit weird as BAP--or Broad Autism Phenotype. If one clinician where to evaluate the parents as having BAP, and then the kid as ASD, that’s like, two weird parents having a super weird kid, that could severely affect the interpretation of the data. Because then you’re not looking for a rare de novo mutation. Then you’re looking for maybe two subtle effect, perhaps common variants, that together compound to a stronger phenotype in the kid. Just, you know, a pitfall of limiting yourself to the rare variant model. Agh sorry, back to the methods.

20 Autism trios.

They did aCGH on them all, and found no large CNVs, except for in one---a maternally inherited deletion--remember? They used trios, so they have data on the mom dad and the kid, important in discovering origin. This is good because there aren't that many great algorithms (yet) to call copy number variations based on exome sequencing results (read depth, etc.) Some groups are working on it, but I don't think it's quite there yet.

Then they did exome sequencing. That is, sequencing the all the exons. (Because that’s the most likely place for a rare protein-altering variant to be, right?)

Filtering methods: They threw out all variants previously observed in dbSNP, the 1000 Genomes Project, and other exome sequencing data they had. The identified <5 de novo candidates per trio, and validated them using Sanger sequencing.

Results

1) The overall protein-coding de novo rate per trio was higher than expected.

2) Using two independent quantitative measures, the Grantham matrix score, for the nature of the amino-acid replacement, and the Genomic Evolutionary Rate Profilng (GERP) for the degree of nucleotide-level evolutionary conservation, they concluded that the de novo mutations they found where subjected to stronger selection and are likely to have functional impact.

3) 4 out of the 20 trios had “disruptive de novo mutations that are potentially causative, including genes previously associated with autism, intellectual disability and epilepsy.”

4) These genes lead to ASD presentation (they go more in depth in the paper, I’m pulling out basically the last sentence of each paragraph that they do.)

a) “Our data suggest that de novo mutations in GRIN2B may also lead to an ASD presentation.

b) “SCN1A was previously associated with epilepsy and has been suggested as an ASDs candidate gene.”

c) “Additional study is warranted, as laminins have structural similarities to the neurexin and contactin-associated familes of proteins, both of which has been associated with ASDs.”

d) FOXP1  encodes a member of the forkhead-box family of transcription factors and is closely related to FOXP2, a gene implicated in rare monogenic forms of speech and language disorder.

Discussion

There wasn’t overwhelming evidence showing excessive burden of mutations in ASD candidate genes. The people that they found potentially causative de novo mutations were all the most severe cases. That is, most had a pretty severe intellectual disability, and features of epilepsy. And also, more importantly, the genes that they did identify with de novo mutations had also been “disrupted in children with intellectual disability without ASD,” to which they acknowledge, “provides further evidence that these genetic pathways may lead to a spectrum of neurodevelopmental outcomes depending on the genetic and environmental context.”

Way to cover your butts, y’all. It might be autism but it also might not.

Here are my thoughts.

First of all, this paper cannot prove causality. Just because someone has a deleterious allele does not mean that that is the cause of the disease that they have. It could be a silent mutation. They could have one good copy. One good copy might be enough to carry you through with no bad affects. Their conclusions of these genes leading to a presentation of autism is purely based on candidate genes and what’s been seen before. The conclusion that de novo mutations may contribute substantially to the genetic etiology of ASD...doesn’t really hold up here, because, well, they haven’t proved that these are the causal mutations.

We know that exome sequencing works in finding new mutations. It’s brought us a long way. This same group brought us the genetic cause of a new Mendelian disease by only sequencing the exomes of 4 people. Exome sequencing works for Mendelian diseases, plain and simple. Therefore, it would be safe to say that exome sequencing also would work for cases of autism that appear to be mini-Mendelian disorders, that is, an ASD phenotype, but a little more severe, so that it happens to be caused by a protein-altering mutation.

However, exome sequencing, and this study in general (again, in my opinion) does not add any new information to the genetic etiology of autism. Correct me if I’m wrong. Did you see a new pathway illuminated? Did you see a really large sample size and a really small p-value? Yeah, there were some pretty looking genes that coded for like, sodium channels, and things that look like neurons, but there was simply not enough numbers, and not enough molecular follow up to claim what they claimed from the start.

In my opinion, and this is truly my own opinion, and sometimes I have opinions on things I’m not professionally trained in but instead on things I’ve read about on the internet, this paper got published because people are so hungry for any inkling as to what causes autism. And their methods, well, are really inoffensive. Sequencing has worked in the past. And it will work again. It will find variants that are rare, and do have severe phenotypic effects, however, it’s not going to help that much with disorders like autism. Or rather, it will help in the cases of autism that act like Mendelian diseases. But being able to explain a large amount of cases that occur, and a lot of the less severe cases that occur? We’re still waiting for that one.