October 10, 2013
Cheap and easy genome sequencing has been both a blessing and a curse. We are able to find an incredible wealth of variation, but for the most part we have no easy way to tell whether a difference might contribute to a disease or not.
The poster child for this problem is autism. Lots of genome wide association studies (GWAS) have been done and lots of rare variants in lots of different genes have been found – unfortunately, way too many to pick out the ones that really matter.
Luckily our friend yeast can help. Various researchers have identified a number of variants in the human cation/proton antiporter gene NHE9 that associate with autism. In a new study, Kondapalli and coworkers used the NHE9 ortholog NHX1 from S. cerevisiae as an initial screen to identify which variants impact the activity of the NHE9 protein. They found that two of the three mutations they looked at compromised the activity of yeast Nhx1p.
They then set out to confirm these results in mammalian cells. When they looked at protein activity in glial cells, they found that all three mutations compromised the activity of NHE9. This is obviously different from what they found in yeast.
Now this doesn’t mean that yeast is useless for this approach (God forbid!). No, instead it means that it is probably only useful for a subset of autism mutations. Kondapalli and coworkers had suspected this, but apparently the subset is smaller than they initially thought.
The first thing they did was to generate a rough three dimensional map of the NHE9 protein in order to see which parts the two proteins shared. The idea is that they could then do a quick screen in yeast with mutations that affect the shared structure.
While the structure of NHE9 has not been solved, we do have the structure of its distant bacterial relative, NhaA. Kondapalli and coworkers aligned the two along with the yeast ortholog Nhx1p and identified conserved regions.
Three of the NHE9 mutations associated with autism—V176I, L236S, and S438P—were all predicted to be in shared, membrane-spanning parts of the protein. The researchers introduced the equivalent mutations into NHX1—V167I, I222S, and A438P.
A yeast deleted for NHX1 grows poorly in high salt and low pH and also has increased sensitivity to hygromycin B, as compared to a yeast with a functioning NHX1. Two of the mutant genes, carrying A438P or I222S, failed to rescue these growth defects. The other mutant gene, with the V167I change, worked as well as wild type NHX1 at rescuing the yeast. So at least in yeast, two of the three mutations appear to impact protein activity.
The next step was to see if the same was true in mammals. Easier said than done! Ideally they would want to investigate whether these mutations affected the protein in the cells where NHE9 is usually active. Too bad no one knows this protein’s natural habitat. This is why the researchers starting slicing mouse brains to figure out when and where the protein is expressed.
While we don’t have time or space to go into all the details here, Kondapalli and coworkers found that when and where in the brain NHE9 was expressed made sense as far as a possible contribution to autism. They also found that glial cells had about 1.2 fold more NHE9 transcripts than did neuronal cells. They therefore did their assays of protein activity in a type of glial cells called astrocytes.
While they couldn’t completely knock out NHE9 in mouse astrocytes, they were able to knock down its expression by over 80%. When they added back the mutant NHE9 genes, they found that all three failed to mimic the effect of adding back wild type NHE9 to these cells. This is different than what they found in yeast, where only two of the mutations impacted protein activity.
When they went back to their 3D model, they saw that the mutation that differed, V167I, affected a less defined part of the structure. This points to the fact that for the quick yeast screen to work, they need to be looking at parts of the protein where the structure is shared between the yeast and the human version. In a perfect world they would have had crystal structures of each to work off of instead of having to kludge together a model.
In any event, this is the first step towards validating yeast as a quick screen for identifying mutations that can impact protein activity and so are good candidates for being involved in disease. Yeast may help scientists separate the wheat from the chaff of GWAS and so help figure out how diseases happen and maybe help find treatments or even cures. Well done yeast.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics