Part IV: Dealing with your Rarer SNPs (2 of 2)

If you're just joining us, you may want to go back to the previous post (link at the end of the entry) because we're continuing our discussion right from where we left off:

Woo hoo! You'll see that rs61750615 "appears to be an incomplete dominant". Lo and behold, there is a citation!

If we click on it, it takes us to an article entitled, Phased Whole-Genome Genetic Risk in a Family Quartet Using a Major Allele Reference Sequence, by Dewey et al, from September 2011 in PloS Genetics.

But when I hit 'search' and type in the rs# I get nothing.

It took me awhile to figure out that the diagrams in the article do not actually get searched. Technically speaking, they're not actually on the same page as the rest of the article; there's a link to get to them. So if we click on each of the diagrams, eventually we get the following:

Oooh, check out the mention of the MTHFR rs1801133, which can contribute to hyperhomocysteinemia, and may add to risk of thromboembolism. That SNP should be on your methylation pages if you got them from MTHFR Support!

Apparently, in the family discussed in the article, the mother, son, and daughter had Von Willebrand disease, and rs61750615 was the only rare SNP they could find on the VWF gene. Using this evidence as well as an algorithm used to predict the hazardous nature of SNPs (the article's main thrust) the researchers conclude that this SNP is what has caused the illness. However, they believe that it is an 'incomplete dominant' trait.

For those of us who remember the TARDIS sound from the first entry, just go along with me and assume we're back in high school biology again. In classical genetics, you have a dominant trait that can 'shout over' the recessive trait. For example, I carry the allele for blue eyes from my mom, but I have my dad's brown eyes, because brown is dominant over blue for eye color. Incomplete dominance is what five-year-olds believe genetics is all about: if trait A and trait B mix, you get a trait somewhere in between. This is like breeding a red rose to a white rose to get a pink one. Or a white-and-red-stripey one. Basically, both traits get to have their say.

The example of incomplete dominance every. Single. Time. :thumbsup:
(Wikipedia, accessed 5/4/2015)

In this case, it could mean that the patients could sometimes produce Von Willebrand factor, and other times seemed less capable of doing so. It could mean that they could produce Von Willebrand factor, but at very low levels. It could mean that they had enough for all reasonable purposes, but if there was a demand for clotting factor (i.e. a major injury), they could start failing to clot. Quite unfortunately, the researchers keep the details to themselves. The article is a genetics article, and does not discuss patient symptoms in detail.

Have you ever noted this? I sure have. When I'm looking after my own health, what's missing from the study is always the perspective of the patient.

Well, here's one, guys: the only time I ever had bleeding issues to speak of was when I had a herniated disc one summer. I also noticed that suddenly little cuts wouldn't heal, and when that time of the month rolled around... let's just say you could cross out 'time of the', and it would be more accurate.

I went to my doc, who sent me to my OBGYN, who took hormone levels and did a physical exam and came back relieved. She said that all my reproductive hormones were totally mid-range normal, and my physical didn't turn anything up either. I'd finally stopped it by pushing Black Cohosh like there was no tomorrow (a dose every 2 hours until the bleeding stopped). But we all know cellular repair was still going on a month later (or you do if you've ever had a herniated disc.) This continued for three months, after which things seemed to normalize. By then my iron and B vitamins were pretty shot, though. After asking me if I'd started passing out or 'losing time' (WHAT.) doc shrugged his shoulders as if it were just 'one of those things'. Like, oh, well! I guess we'll never really know! (Yep - it's the same fool from earlier.)

I've also noticed that my clotting rate is pretty variable, but never much to my detriment, otherwise. If I cut my finger, I've noticed sometimes it'll clot immediately, and other times I'll look at it an hour later and it'll just be closing.

So, let's say this is the only information you can get your hands on. (In my case, it is.) What else is there to do?

If you have a strong suspicion that you may have a particular condition due to a certain SNP, remember that the SNPs shouldn't be taken as the final word on anything, but used as a direction for future study and analysis. Getting a blood, urine, or stool sample analyzed for the disorder in question is the next step, if that's a possibility.

For example, Von Willebrand's Disease is diagnosable via blood tests. Unfortunately, even people who do have it (non-partially-dominantly) can sometimes come up with numbers in the normal range if they haven't bled a lot lately. Even the more official looking sites suggest you do it again three months later if it comes back negative and there's evidence of the disorder. I'm deciding whether it's worth the time, money, and energy to hunt it down like a wildebeest of the veldt.

[Edit, 3/28/16: This bleeding issue may very well be due to 'anemia of chronic infection'. After noticing that my clotting and bleeding issues seem worse when my overall illness seems fiercest, I began looking into this theory. The body produces anticoagulants in infection, which makes sense in terms of trying to clear out a wound. Discussion and citations in this thread.]


Phased Whole-Genome Genetic Risk in a Family Quartet Using a Major Allele Reference Sequence
Dewey FE, Chen R, Cordero SP, Ormond KE, Caleshu C, et al. (2011) Phased Whole-Genome Genetic Risk in a Family Quartet Using a Major Allele Reference Sequence. PLoS Genet 7(9): e1002280. doi: 10.1371/journal.pgen.1002280

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<----Start the series


We can't write long responses to blogs, so here's part I:
There is a way to predict the impact of a missense mutation - perhaps similar to what the non-forthcoming researchers used :rolleyes:

The simplest is to use a substitition matrix chart. BLOSUM62 seems to be the most common. Basically the higher the score when substituting one amino acid for another, the more similar the two amino acids behave and it's less likely that the substitution causes serious problems. The negative values are for the amino acids which behave pretty differently, with -4 being the worst on the BLOSUM62 and 3 being the highest possible value for a substitution.

So your Von Willebrand missense mutation results in proline being replaced by serine. On the chart at that gets scored at a -1. So not a very low value, but not a high value either. Again, rather disappointingly inconclusive!

Fortunately there's more complex modeling software which can make use of a great deal of data, including 3D modeling and predictions of structural changes and the impact which they might have . The one I know of is at and requires setting up a free account with a valid email (sometimes it takes a long time to process, so results are emailed in those cases). After logging in, you can go to the link I just listed, or click on "Run sapred_seq" from the left side. Then you click on the "+" box in front of "input FASTA file". The FASTA is basically the structure of the protein, in a specific format.
Part II:
To get the FASTA sequence, you can go to and search for the gene by it's proper abbreviation - we can get that data from the dbSNP page where we see the other data for your rare SNP. So it's called VWF, and you can just put that into the search bar at the top of the Uniprot page. Then it's very important to look for "HUMAN" in the search results - mouse is listed first in that case, which could cause a lot of confusion if selecting that instead :D

So that should mean you're clicking on entry P04275 at uniprot. There's a ton of interesting data there, but to get straight to the FASTA data, you can either scroll down quite a bit or just click on "Sequences" on the left to get there faster. If there's more than 1 sequences, you want Sequence 1. Under the "Sequences" section, there are several blue boxes ... the one with a down arrow and "FASTA" is the one you want, so click on that.

This brings up a new page with some gibberish and a huge string of letters which represent amino acids, in the order which they appear in the VWF protein. This happens to be a pretty damned long one - most are much shorter. Practically speaking, this means that processing time will probably take longer than 30 seconds, and sapred will email you when the results are done, or you can just check back every minute or two, because it will give up on showing you the immediate results after 30 seconds of processing.

Anyhow, you need to copy the entire text appearing at . Then we go back to the sapred page, which is hopefully still open in your browser, and paste it in to the right of where it says "OR paste into window".
Part III:
Next you need to fill in the "mutation name" in the little window right under where you pasted the FASTA sequence. You can get that data from the dbSNP page for the SNP. Basically it's always right above the map view, with some red text under "Residue Change". We need both the normal amino acid, plus the protein position, plus the mutated amino acid, so the format should end up looking like P2063S. And that's what we put into the sapred box next to "mutation name" :)

(This previous step can get complicated when dbSNP lists numerous positions for the same SNP. Only one of them is right, which can be figured out by going back to the "sequence" at uniprot and seeing which of the listed positions actually has the "P" or other relevant amino acid. Sometimes this still results in multiple possibilities, and you need to read the adjacent codons at dbSNP to figure out what the next amino acids are, so you can confirm which position is correct for your SNP.)

Now that the FASTA and mutation name have been filled in at sapred, it's time to click "Run". And now we wait, probably for several minutes because the VWF protein is so friggin' big ... Yup, it's telling me to either wait for an email or check the "history" link on the left periodically. The history shows me that the status of the most recent job at the top is "Running." So now I wait more, and check again ...
... and now clicking History yet again shows me it's Finished. Yay! And FYI, it took 150 seconds. My record so far is 664 seconds for the ABCA4 gene protein.
Part IV:
Anyhow, now you need click on the link for "OUTPUT FILE". This first shows you the FASTA sequence you entered, then you can scroll down in the box and it'll also show the mutation name, and finally the results! The prediction is "Neutral" which means that it's more likely that it doesn't have a major impact. In this case, it's a 59.6% likelihood that the mutation is neutral. It's far from conclusive (the highest possible values seem to be in the upper 80's or low 90's), and will never be certain since it's assessing probabilities, often with limited data. But it's probably not doing much.

You can also go back to the main Uniprot page for the VWF and look at the positional information. The "Pathology and Biotech" section indicates it's a "Natural Variant" and doesn't seem to have any data on it beyond that. The next section is also interesting, PTM/Processing. Under the "Amino acid modifications" subheading, you can see if your mutation is in one of the more important parts of the protein, such as where it's attaching to other proteins, or forming internal bonds. There's nothing listed at 2063, so probably the only effect that mutation would potentially have is from breaking down a bit faster or slower, due to the substituted amino acid forming stronger or weaker links with its neighbors than the original amino acid does.

But it's not looking like a likely problem currently. And the research is listing the diseases which can be associated with the SNP's gene, rather than listing diseases affecting the family members. The only disease they're known to have is familial thrombophilia, and that's due to a known mutation which they have on the F5 gene. I also don't see any citation to something which might indicate that that particular SNP is implicated in causing any problems.

So that study found that the mother and children have a rare variant on VWF, but no apparent symptoms of that disease.
Woowww, Valentjin! I had seen programs like that. I am so impressed by your tutorial! I'm going to try this later today! :D
Registered for an account, Valentjin! Now waiting... is it all right with you if I end up writing this up as an additional post? I will credit you, of course, if I do.

And saying 'no' is totally okay. :)


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