Saturday, May 31, 2014

Rebuttal to "Genetically Modified DNA transfers from food to blood" (or "How Bt corn can cause the Zombie Apocalypse")

This is part 2 of a series looking at papers that have used next generation sequencing (NGS) technology, which are used as examples of how eating DNA from a GMO could be harmful. If you missed the previous post, you have to go back and read it. There are a lot of cookie analogies in here that will be completely lost on you unless you've read it. If you HAVE read it, make sure that you have some chocolate chip cookies in the house. You may find yourself craving some.

The first paper that we’ll examine is entitled "Complete Genes May Pass from Food to Human Blood". The paper was published in July 2013 in PLoS One, and is highlighted in this article from Collective Evolution entitled "Confirmed: DNA from Genetically Modified Crops can be Transferred Into Humans Who Eat Them" (the graphic in that article is nightmare-inducing). In this paper, they examined the content of DNA outside the human cell, known as "cell free DNA" or cfDNA. As a reminder, the DNA we inherit from both our parents is packed up nicely and tucked away within the nucleus of the cell. The paper outlines that the source of DNA in our plasma (i.e. the stuff that's in the space between our cells) is thought to originate from cells that have died. However, there are also foreign sources of DNA in plasma from bacteria, viruses, and from our food. Fetal DNA can also be detected in maternal plasma and is the basis for non-invasive prenatal testing (NIPT).

File:AntiMonsanto March GMO Corn.jpg
March Against Monsanto
New Orleans, May 2013
The authors of the paper took 200 blood samples from 4 different types of patients who had different intestinal diagnoses, and included patients with no symptoms (i.e. negative control). They separated the blood from the plasma, they extracted the DNA and they pooled the DNA from each group. So, for example, if there were 50 patients with irritable bowel syndrome and 50 control patients, each group of 50 was pooled into a single tube so that there were only 2 samples at the end: one sample representing the irritable bowel syndrome patients and another representing controls. I can only think of two reasons why they'd do this: 1) sequencing each individual patient was too expensive (next generation sequencing is pretty pricey) or 2) they didn't have enough DNA since there's so little floating around in the plasma. I'm leaning towards #2, because they also concentrated the sample (i.e. removed water content so that there was more DNA in less liquid). Then, they sequenced the pools of samples using next-generation sequencing technology (for the NGS gurus, they used SOLiD with 50nt reads).

The authors threw out all the DNA sequences from vertabraes because a) they weren't interested in human DNA sequences and b) it would be difficult to tell what organism the DNA came from due to similarities in DNA sequences (after all, we're more similar to chickens that we'd like to believe). Then they took the remaining DNA samples and compared them to a database of sequences of chloroplast DNA. Chloroplast DNA is unique because it is separate from the DNA found in the nucleus of the cell. It is circular and there are multiple copies of chloroplast DNA in each plant cell (sounds a bit like mitochondrial DNA, if you're familiar with that from 23&Me and other ancestry DNA sequencing services). The authors found that there were quite a few sequences that matched chloroplast DNA, particularly the DNA sequences for potato and tomato chloroplast, and actually got more data of tomato DNA than human DNA in some regions.

Then, they wanted to determine the original size of the DNA fragment. It is generally thought that most DNA gets fragmented during the digestion process, so if they could demonstrate that the DNA that was sequenced was long, then you might be able to make a case that entire genes could be floating around. However, this is pretty difficult to do because during the process of preparing a sample for next-generation sequencing, you generally chop up the DNA into bits and pieces. If we go to a cookie analogy, imagine that you make chocolate chip cookies with walnuts. You buy a bag of walnut pieces, which may contain a few whole walnuts. The recipe calls for throwing the walnut pieces into the food processor before you add them to the cookie batter. So it's pretty tough to figure out how many whole walnuts were in the bag by eating the cookies.

To get around this conundrum, the authors physically filtered the DNA according to size. They had 3 filtration sizes which became 3 different samples. Each sample was then chopped up and when it was sequenced, you could infer that the DNA's original size was larger than the filtration cutoff (for the science-y people, they ran a gel and cut three bands from the smear: >10kb, 10kb-200bp, and ~200bp). If we go back to our walnut analogy, imagine that you take the bag of walnut pieces and pass it through a 1/2 inch sieve. Everything that gets caught goes in one bowl. Then you take the stuff that went through and you pass it through a 1/4 inch sieve. You repeat the process with a 1/8 inch sieve. Then you take the 3 bowls of walnuts and you put each one of them through the food processor, make the cookie batter, and end up with 3 batches of cookies. All 3 batches will have roughly the same walnut size, but you can infer that the original starting size of the walnut pieces was >1/2", 1/2-1/4", and 1/4"-1/8" (BTW, I honestly don't understand this whole Imperial measurement system. The Canadian AND Venezuelan parts of me are shuddering as I write this).

The authors infer that a lot of DNA sequence came from the largest filter size from patients diagnosed with irritable bowel syndrome (IBS). The filter size that they used was 10 kilobases. If you consider that the average size of a human gene is 10-15 kilobases, then this implies that most of the cell-free DNA in patients with IBS is large enough to have a gene in it.

The authors then wanted to confirm their findings. They searched publicly available DNA databases and found 909 samples of cell-free DNA, representing 907 individuals. They also found non-human DNA in the electronic data, but noted that the amount that was present had "large variations" from person to person. They followed the same data analysis workflow as before. The DNA in the public databases came from 2 projects: one project was studying patients with an autoimmune disorder and the second was trying to detect fetal DNA in pregnant women. Here's the breakdown of the DNA from the two studies.
  1. Autoimmune disorder: The most common matches were to chloroplast DNA from Brassica rapa, as well as orange. The machine used to sequence these samples was not the same as the one used by the authors. The authors state that there's a lot of plant DNA in these samples when compared to control. Since this is the same observation noted in the patients with irritable bowel syndrome, the authors state that high levels of plant DNA circulating in plasma may be associated with inflammation. I'm holding my tongue on all criticisms of this paper till I'm done with the description, but I can't help myself from saying "wha-aaaat? how did you jump from here to there???"
  2. Pregnant women: There wasn't much sequencing data from these samples, but the authors were able to determine that the most common match to chloroplast DNA were from soybean. Additionally, since these samples weren't actually pooled together (i.e., each sample was sequenced independently), the authors were able to identify differences in the abundance of plant DNA in these samples, which represents differences in the diets of the pregnant women. For example, if I had been a participant in this study, I have no doubt that the authors would have identified an abnormally high level of chloroplast DNA from pomegranates. This finding suggests that the plant DNA detected in these samples are not actually contaminants.
The authors conclude that the presence of foreign DNA in the plasma is not unusual, that its concentration is highest in patients with inflammation, and that these findings should lead us to revisit our views on the degradation and absorption of DNA/RNA in our bodies.

I think that the finding that there is plant DNA circulating in our bodies isn't a big deal. The paper provides several references for studies that have examined this issue and have found DNA from our food in our organs and tissues (see here and here). However, it's always been chopped up. This paper suggests that full genes are floating about, which is what raised the alarm flags for activists. So I'm going to focus on this unique finding from the paper. 

Getting back to the paper. I have several issues with the experiment the authors performed in their lab (i.e not the data analysis work on the plasma samples from autoimmune disorder patients or pregnant women):
1) Contamination. As I stated at the beginning of this piece, the authors are sequencing the DNA in the space between our cells. There's very little DNA in there so the risk of sequencing a contaminant is high. To recap from last week's piece, it's a matter of abundance: if you had actual cellular material, all that plant DNA would get drowned out by the vast amount of human DNA that you'd end up sequencing. As mentioned last week, like having 1 cup batter of chocolate chip-raisin cookies with a handful of cranberries that your kid threw in versus 1 gallon batter of chocolate chip-raisin cookies with the same amount of cranberries. Since the authors probably had very little DNA when they started, any DNA from the environment or from their equipment could be mistaken for DNA from their samples.
Since the risk of contamination is higher, the authors should have included a negative control. Going back to the cookie analogy, to determine if the cranberries are part of the chocolate chip-raisin mix in the cookies or not, there's a very simple test: make a batch of cookies with no chocolate chips or raisins. If you end up with cranberries in there, then you can conclude that the cranberries are a contaminant (i.e. your kid walked by and threw a handful in there). If there are no cranberries, then you can conclude that the cranberries were part of the chocolate chip-raisin mix. The authors failed to do this simple test.
I was happy to see that this point is also noted in the comments section by a scientist who has published a rebuttal. I'll review this further below. 
2) The authors find high levels of tomato and potato DNA in all their samples. This doesn't make much sense to me. Why would the authors find the same two DNA samples to be of highest abundance in all the different patient types and filtration sizes? As seen in the study with pregnant women, there should be variation between the different groups. I know that tomatoes definitely don't make up the biggest part of my veggie/fruit diet, so this is really weird.
3) The authors find abnormally high levels of plant DNA in the irritable bowel syndrome patients, but only for the largest filtration size. The authors conclude that foreign DNA in plasma is elevated in patients with inflammation. As such, you'd expect to see increased levels of foreign DNA in every filtration size. However, the medium and small filtration sizes have plant DNA levels equivalent to the patients with no symptoms. There's one thing that I think you can agree with: concluding that "plant DNA is elevated in patients with inflamation" is a HUGE conclusion to draw from a single sequencing run.
4) Ummmmm... Filtration controls? Where are you? The authors infer DNA size based on physical separation of DNA. However, they have no controls. It would be fairly simple to just spike in DNA of different, but known, sizes (the use of a "ladder" in DNA size separation is very, very, very, very, very common, so it would have been trivial to do). This size control would have also helped determine contamination: if you find some of the large DNA control in the small DNA results, then you know that some sort of contamination may have occurred during the filtration process. It would be similar to placing a brazil nut, a hazelnut, and a peanut whose sizes you've measured into the walnut size separation. The brazil nut should filter out with the large walnut chunks, the hazelnut with the medium chunks and the peanut should end up in the small bits and pieces. If any pieces of these nuts appear in the "wrong" cookie batch, then you could conclude that there was contamination. Maybe you didn't wash the blade on your food processor well enough. Or maybe you got carried away by the music you were playing in the kitchen and made an inadvertent mistake. Seriously. Anything is possible, and if you don't have controls, you'll never know. 
5) Choice of NGS technology. As I mentioned in the first installment in this series, different NGS companies have different chemistries, all of which have pros and cons. The technology that the authors of this study chose required the DNA to get chopped up to small bits and pieces, leading them to infer that the DNA was long, but not measuring the length directly. They didn't have to use that specific chemistry. I would have chosen a technology that would have allowed them to sequence longer lengths of DNA (for the NGS geeks, I think that PacBio might have been a better fit). It depends on how much DNA they had to start with, and they don't really elaborate this point. However, given the fact that they pooled together DNA from 50 patients, I think it might have been possible.
6) Why chloroplast DNA? I think it's odd that they focused exclusively on the  analysis of DNA from the chloroplast, and not the DNA from the nucleus of the plant cell. Is this truly reflective of all the DNA in the cell? Is it possible that due to the circular nature of chloroplast DNA, it can avoid degradation more readily? Since there are more copies of chloroplast DNA in each cell, how does this affect their findings?

But, let's imagine that the findings of the paper are not an error and that someone else actually replicated these findings. What does it mean?

  • This has little to do with GMOs. I feel the need to reiterate that if a full gene for a transgenic food is floating in our system, so is a full gene from a traditionally bred crop. Additionally, scientists haven't gotten smart enough to invent new genes/proteins, so whatever gene is in a transgenic crop, also comes from nature. The only difference is what you ate in order to get that gene into your system. This fact alone should debunk titles of articles such as "Genetically Modified DNA transfers from food to blood" (also, now that you've gone through this post, you know that it's not actually blood that was studied here)
  • As I said at the beginning of this post: then what? Somehow these whole genes that are floating about have to make their way through the outermost layer of the cell (cell membrane), avoid getting degraded by proteins that chop up foreign DNA, and make their way into the nucleus. Within the nucleus of our cells, it would then somehow have to "trick" regulatory proteins so that they think that the foreign gene has to be turned on, so that it gets made into RNA. An alternate option is for the foreign DNA to get integrated into the cell's DNA (i.e. act like a virus), even though it doesn't have any of the viral proteins/genes. But let's say that somehow one of these scenarios were to play out, and the gene that was floating about was the transgenic gene from a GMO corn (the odds of this alone are 1 or 2 in 32000, since there are only 1-2 transgenic genes added to corn, which has 32000 genes), and that this DNA somehow managed to defy all odds and get made into RNA. The RNA will then be made into a protein. And let's pretend that this happens stably: meaning that this protein keeps getting made. That's 1 cell out of the 46-68 trillion in our body that is making a foreign protein. The two most likely fates for this protein produced by this single cell in your body is a) your immune system will take care of matters or b) the protein will just fade away (all proteins have a half life; they don't just float around forever). If you want to lose sleep over that, go right ahead.  I'm more worried about the zombie apocalypse, and the CDC thinks you should be too.
There has been a rebuttal paper written to the PLoS paper, however, it is in peer review and has not yet been published, so I'm hesitant to add its findings. I'll update this post once the paper is accepted.

Well, that's paper #1. Next week, we'll review a controversial paper that found that small RNA from rice can regulate a protein in our bodies and all the subsequent papers that attempted to replicate the findings.

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