N24 Awareness Day 2017: Through the Looking Glass

24 November 2017 at 07:34 | Posted in Body clock, Circadian rhythm, Clock genes, N24 | Leave a comment

I apologize if this year’s post is shorter than most. I am still recovering from a nasty case of the flu which has really drained my energy. But N24 Day must go on!

Many of you reading this post will have some idea of what N24, also known as Non-24 or Non-24-hour sleep-wake cycle disorder, consists of. But as the theme for today is awareness I will first start off with some links that explain the disorder.

I have written an article for Sleep Review entitled All You Need to Know about N24, which many patients and health professionals have found helpful. I also co-authored (with Dr Katherine Sharkey) a report on Non-24 for the the National Organization of Rare Disorders. Another excellent source of information on N24 and other circadian disorders is the Circadian Sleep Disorders Network. If you have a circadian disorder please take their survey.

The theme of this year’s N24 Awareness Day is Through the Looking Glass. This theme is meant to reflect the strange time-disordered world those of us with N24 find ourselves living in. Even Lewis Carroll did not imagine the strangeness of creatures living on a 25 or 26 or 30 hour cycle in a 24-hour world. (Although one may wonder if the White Rabbit was one of us, always running late and looking at his watch.)

But I want to talk about something that happened this year which may be a through-the-looking-glass moment for the science of circadian rhythms and the public awareness of the same. The 2017 Nobel Prize in Physiology or Medicine was awarded to Jeffrey C. Hall, Michael Rosbash and Michael W. Young for their discoveries of molecular mechanisms that control circadian rhythms.

Starting in the 1980s Hall, Robash and Young worked on the oscillation of proteins in the fruit fly and with an amazing amount of painstaking work over many years were able to draw a picture of what, in a biochemical sense, are the gears of the internal clock. The clock in mammals, such as humans, turns out, with some modifications, to work the same way as in fruit flies.

The basic model is that of the transcription-translation feedback loop. Regions of DNA are transcribed (copied into RNA) and then translated (the RNA is used to make protein). The proteins produced then act to control the transcription/translation process that produced them. As more of the end protein are produced signals are sent back to say, “make less protein” and so the level starts to fall again. A rise and fall of specific protein levels occurs in a rhythmic fashion, and numerous other loops of protein transcription/translation help to fine-tune this rise and fall so it occurs every 24 hours in a healthy organism.

The key protein in Hall and Robash’s early work was the PER (for period) protein. Young then discovered another gene/protein combination, the timeless gene producing TIM protein. Together these form the basic parts of the fruit fly clock (but many other parts are needed to make it work precisely). Other researchers then extended this work to mammalian (including human) clocks, discovering the roles of the CLOCK, CRY and other proteins.

This research has been going on for many years, so what I have been describing is not new. What is new is the decision of the Nobel Prize Committee to give this work the recognition it deserves. This is part of a trend–more and more papers at sleep conferences and other scientific societies now focus on circadian rhythms.

For too many scientists, doctors and members of the general public it has been tempting to think of circadian rhythms as something secondary, something superimposed on the basic functions of the body, but which one can generally ignore. This view is no longer tenable. The transcription-translation feedback loop that controls the circadian clock lives in the heart of every cell in the body, literally inscribed in the DNA that allows us to live. Those who pay attention have realized this for some time. But the Nobel prize is a bell in the dark, alerting the rest of the world to this fact.

Those of us with N24 are not the sole inhabitants of the mysterious beyond-the-looking glass realm of circadian rhythms. That realm is the inner sanctum of life itself. We live on a planet that spins every 24 hours and this is imprinted on our being.

This prize is not going to mean a sudden era of rainbows and sunshine for those of us with N24. It may be years before these discoveries will have practical impact on our condition. But this is nonetheless a big step. When we say to our doctors or families or others that we have a circadian disorder, we can know that the Nobel Prize committee has told the world, in no uncertain terms, that circadian rhythms are real, they are important, and to them attention must be paid.

James Fadden

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N24 Awareness Day 2016: Genetics, the new face of N24 and DSPS.

23 November 2016 at 05:26 | Posted in Body clock, Circadian rhythm, Clock genes, DSPS, N24 | 4 Comments

I got my genes tested this month, and what I found out puts a new face on my understanding of N24 and DSPD.

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The first human genome was sequenced in 2003 and cost 2.7 billion dollars.  Over the years researchers have improved their technique and reduced the cost dramatically.  Recently Veritas Genetics announced it had reached the goal of the under $1000 genome, offering a full genome sequence to anyone for $999.  Still very expensive but quite a drop from 2.7 billion!

Other companies also offer a more limited but still useful form of genomic testing.  One of these is 23andMe which offers testing for $99-$199.  Rather than plunging into the deep end  of whole genome sequencing, I thought I’d go with 23andMe to confirm that I could get useful information with less expense.  I am very impressed with what I was able to find out from 23andMe.

The method of 23andMe is based on analysis of what are called single nucleotide polymorphisms (SNPs).   Most of the human genome is identical from person to person, which is why we are all humans and not horses or banana trees.  SNP analysis focuses on variations in the genome of a particular common type.

The genetic code is a sequence of DNA nucleotides which we label with the letters A,T,G or C.   So part of the code might read like this: …CTGAATGCAGT…  An SNP refers to a situation where one letter of the code differs from person to person.   So while one person may have the sequence CTGAATGCAGT  another may have the sequence CTGAATTCAGT.   Notice the only change is that the letter G in the 7th place has become a T.   Typically one SNP will be more common in the population than the other.  So, for example 90% of the population may have a G, which would be referred to as the major (more common) allele.   The T would be called the minor allele.  (Allele is another word for genetic letters.)

So if you sign up for 23andMe you send them a vial of your spit, and 4-6 weeks later they send you a link to their site where they give you information about a selection of your SNPs and what they mean, as well as some other genetic information.

Most of the information you get from 23andMe, at first glance, seems pretty basic.  They tell you where your ancestors came from and a list of various genetic traits: is your hair likely to be curly, can you taste bitter foods etc.  Interesting but hardly world-shaking in most cases.

But in addition to this pre-analyzed information 23andMe also allows you to download a file containing the raw data: a long list of the actual SNPs and your results.   These you can upload to certain web sites such as Promethease for detailed analysis, or if you know what you are doing and what you are looking for you can look through the raw data yourself.  That’s when the fun begins.

And I knew just what I wanted to look for.

In 2014 Daniel Kripke et al. published an article called, Circadian Polymorphisms in Night Owls, in Bipolars, and in Non-24-Hour Sleep Cycles.  You can get the full text at the link below.

https://synapse.koreamed.org/DOIx.php?id=10.4306/pi.2014.11.4.345

The study identified several SNPs statistically associated with N24 or DSPS.   Not all of the SNPs studied by Kripke et al were tested by 23andMe but three of the most important ones were.

In the case of N24, Kripke et al. were particularly interested in variations in a gene called BHLHE40, or basic helix-loop-helix protein E 40.   The protein produced by this gene plays a major role in the molecular clock.

http://www.uniprot.org/uniprot/O14503

The study found that subjects with N24 were statistically more likely to have one or two C alleles instead of a T at a portion of the genome labelled rs908078, which is part of the regulatory sequence for this gene.   Looking at my 23andMe data I found this line:

rs908078        3       5024771 CT

So, yes indeed I did have one C allele.  It might have been even more impressive if I had two, as some of the N24 subjects did, but one C is still interesting.

But I developed N24 as an adult, following chronotherapy. Before that I had DSPD.  So if there is a genetic predisposition it might be even more likely to show up in genes associated with DSPD.  Kripke et al found two such genes and corresponding SNPS.

The first is a gene called NFIL3 (nuclear factor, interleukin 3 regulated).  It also plays a role in the circadian clock.

https://www.ncbi.nlm.nih.gov/gene/4783

The SNP for NFIL3 is rs2482705, and people with DSPS are more likely to have two G alleles. So looking at my data file I find this line.

rs2482705       9       94182502        GG

So, yes, GG.  I can cross out another letter on my bingo card.

The other gene is RORC (retinoic acid receptor-related orphan receptor C).  This gene has many functions and is not well understood, but one of its roles is also in clock regulation.

http://www.genecards.org/cgi-bin/carddisp.pl?gene=RORC

The associated SNP is designated rs3828057.  People with DSPS are more likely to have a GG allele.  Going back to my data file I search for that string and find this.

rs3828057       1       151780177       CC

You might at first think that was a miss, but remember the structure of DNA.  It consists of two strands linked together in a helix, which run in opposite directions, the sense strand and the antisense strand.   A C in the sense strand matches a G in the antisense strand, and vice versa.  An A matches to a T.  So CC is actually equivalent to a GG in this case.  It simply means one group tested the sense strand and the other the antisense strand.

So we have another hit.

So for the SNPs that were tested  by 23andMe I am 3 for 3 in having the alleles associated with N24 or DSPD.  I don’t want to make too much of this.   These are statistical associations.  It’s entirely possible to have either disorder and not have these genes or to have the genes and not the disorder.  Nonetheless, while I am not yet ready to shout BINGO!,  I find the presence of these genes intriguing.  We aren’t quite ready to trace their function directly to the disorder but that may come in time. The first step in that process is to know what genes are involved. The fact that these genes are ones we do know are intimately involved in regulating the circadian clock is a good omen for our future understanding.

But there is also more on the horizon.  Recall I mentioned 23andMe only tests a limited number of SNPs.   Kripke et al reported a total of 9 SNPs associated with N24.  While rs908078 was the one they focused on the most, the other 8 are also significant.  But 23andMe only tested for rs980078.  They did not test the other 8.  But a whole genome sequencing, if I am ever able to afford that, should give results for the other 8. That’s a lot more letters to put on the bingo card!

I don’t think we are likely to explain N24 or DSPD entirely based on genetics.  Developmental and epigenetic factors almost certainly play a role.  But the more we know about the genetic aspects, the better off we will be. I must also add that genetic studies are not always replicated and it may turn out that all of this is a Will-o’-the-wisp that I will have to retract next year.  But eventually real data will come out.  As one of my favorite fictional characters said, “the truth is out there.”

LivingWithN24 (James Fadden)

This post also appears on the CSD-N web site.  Please join CSD-N to help support research like this.

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