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 | 3 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.

60. Charting the course of N24

27 October 2010 at 09:25 | Posted in Circadian rhythm, DSPS, Sleep diary | 27 Comments
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Keeping a sleep log or a sleep chart is one of the keys to getting a proper diagnosis of a circadian sleep disorder.  Oftentimes someone will describe their sleep patterns, but it’s not always clear from the description when exactly they sleep.  For example, someone will say they “keep going to bed later and later.”   Depending on what they mean, that could either describe Non-24 Hour Sleep-Wake Cycle Disorder (N24) or a gradually worsening case of Delayed Sleep Phase Syndrome (DSPS).    But a quick look at a sleep chart will usually clear up the distinction.

My eponymous co-blogger Delayed2Sleep, who has, of course, DSPS, has posted several of her sleep logs.   Today I am going to post some of my sleep charts.  My name is LivingwithN24 and I have, you guessed it, N24.

One thing I should note to start is that I have always charted my sleep on a vertical chart, whereas D2S has used a horizontal layout, so you’ll need to rotate the charts to compare them.  I have also “double-plotted” some of my sleep charts.  That means each line on the chart encompasses 48 hours.  That’s a standard method in sleep studies as it helps the patterns to stand out more clearly.

I have had N24 for many years.  Here are two pages from a chart of my sleep I compiled in 1991.   In those days I kept my charts on graph paper.

These charts show a textbook case of N24.   The phrase “textbook case” is not a metaphor here; the medical journal articles about my case are commonly cited in reviews of circadian sleep disorders.  So there is no doubt you are looking at a case of N24 sleep.  One can see that the sleep chart of an N24 shows a characteristic diagonal pattern as the bedtime and waking time slides gradually later each day.  Unlike the case of DSPS, the delay does not stabilize at a particular clock time.  Someone with DSPS may for example end up going to bed always around 6am for an extended period.   Someone with N24 who goes to bed at 6am on Monday will likely go to bed at 7am on Tuesday, 8am on Wednesday, 9am on Thursday etc. until  they have gone all the way around the clock.  The delay is not always by one hour — I just chose that for simplicity.  In my case I tended to delay 1.5 hours per day, meaning my “day” was actually 25.5 hours.

One can see in this chart that there is some irregularity in the sleep times, and there may also be daytime naps.  Both of these showed up a lot in January and February of that year.  But there is a nonetheless a clear pattern to the sleep.  In some months such as March, April and May it stands out especially clearly.

It might also be of interest to compare these charts to another type of chart.  In circadian medicine a technique called actigraphy is used. The patient wears a device called an actimeter on their wrist which records movement.  Since people tend to move about a lot more when awake than when asleep, actigraphy can be a way of confirming the sleep times of a patient with N24 or DSPS.  Here is an actigraphic chart of my sleep from around the same time as the sleep logs.   The difference is the black squiggles on the actigraphic chart show time awake, rather than asleep, but the same diagonal pattern is present.

Now those charts are from 1991.  The readers of this blog are clever folks and have no doubt noticed it is now 2010. I did this for a reason.  Most of the studies of N24 have looked the subjects at one point in their lives.   Few, if any, have examined how the sleep patterns of N24 change over the course of many years.  I have noticed a number of changes in my sleep between 1991 and today.

One thing I noticed is that it is much more difficult to control my N24 with lights, darkness and sleeping medications.  In fact, over this summer it became impossible.  So in September I stopped using the lightbox and the sleeping pills to see what my sleep would do on my own.

Here is a sleep chart I made for the last 5 weeks (Sept 18-Oct 25, 2010).  Instead of making the chart by hand I used the SleepChart program from supermemo.com.

This is quite different from the 1991 charts.  While there is still a recognizable diagonal drift of the sleep times, it is much more erratic.  The times of sleep are much less regular.  Also there is a marked tendency to show two or more periods of sleep instead of one for each circadian day.

In some ways this pattern of sleep is in between that of Non-24 Hour Sleep Wake Cycle Disorder and that of Sleep Wake Cycle Disorder – Irregular Type.   In the irregular type of sleep disorder, the person sleeps at almost random times of day or night and for varying lengths of time.  Usually there are several short periods of sleep scattered at various time around the clock.

My sleep has not become completely irregular, but it is getting there.  This is, to put it mildly, not a good thing.  N24, bad as it is, has a least a little predictability to it.  I could never predict my sleep out several weeks as some very regular N24s could do.  But I could at least predict it fairly well 5 or 6 days out.   Now I can barely predict what it will do from one day to the next and the urge to sleep or be awake comes upon me at almost random times.

Usually irregular sleep patterns are associated with conditions involving brain damage (injuries, tumors) or deterioration (dementia).   However I suspect that in cases of severe circadian disorder the circadian system may deteriorate over time, leading to an evolution towards an irregular sleep pattern.   My co-blogger has remarked that her DSPS sleep has some irregular elements as well.

Instead of a static snapshot of sleep which classifies the disorders as DSPS, N24 or irregular, research needs to look at sleep disorders as dynamic, evolving conditions.  If someone has say, heart disease, we understand it will manifest itself differently at age 20 and 40 and 60.  The same may be the case with circadian disorders.  I had some sleep abnormalities from birth, classic DSPS from age 15 to 27, and N24 from then on.  Now I seem to be trending towards irregular sleep.   My approach to coping with this condition will have to change as well, but how that will happen I don’t yet know.

–posted by LivingWithN24 (James Fadden)

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Next post:  61.  Questions and answers about Non-24

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55. Chronotherapy: balancing benefit and risk

2 August 2010 at 15:16 | Posted in Body clock, Circadian rhythm, DSPS | 18 Comments
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Treatments for circadian sleep disorders fall into three general categories. The first combines phototherapy and scototherapy, that is treatment with light and dark.  The second is pharmacotherapy, usually using melatonin or one of its drug analogs. The third treatment is what I will discuss today and is known as chronotherapy.

None of these forms of treatment is universally successful — or there would be little need for this blog.   Chronotherapy was the first treatment found to  be successful for some cases of DSPS and thus was a major advance in treatment [1].  The researchers who discovered it should be commended.  But many valuable medical treatments also carry inherent risks.  In particular anyone planning chronotherapy needs to know that it poses a risk of converting DSPS into the even more severe disorder known as non-24 hour sleep wake cycle disorder (N24).

This risk was first reported in a letter to the New England Journal of Medicine in 1992 by Dan Oren and Thomas Wehr of the NIH [2].  They described three patients who had long-standing DSPS and had attempted to treat it by means of chronotherapy. In each of those cases the result was a persisting case of N24.

I can vouch for the accuracy of the NEJM article for one of the cases described is actually mine. I had DSPS for over 15 years before attempting to treat it by chronotherapy.  That was the start of my current condition of N24.

The reason I am posting about this at this time is that I have gotten emails from people in recent years who have tried chronotherapy and who had also ended up as N24.  These  people were startled to find out that this risk was known 18 years ago. They were not aware of this risk prior to starting chronotherapy. Since chronotherapy is widely recommended, but the risk it poses is not widely known, I thought the subject needed to be addressed.

Two questions arise.  Why does chronotherapy cause N24 in some cases; and how often does it do so?

To address the “why” question, let’s first review the difference between DSPS and N24.  Someone with DSPS is unable to sleep except at a very delayed hour compared to most people.  For example someone who sleeps every day from 4am to noon and cannot advance their sleep to normal hours would have the diagnosis of DSPS.

N24 is somewhat different.  The sleeping time of someone with N24 changes from day to day. If they start out falling asleep at 4am, the next day they might not fall asleep until 6am, the following day at 8am, then 10am and so on, until they go around the clock. They might have a 26 hour day, as in that example, or any other day length longer than 24 hours, hence the name non-24 hour sleep-wake cycle disorder.

Returning to DSPS, while someone with DSPS cannot advance their sleep — cannot start going to bed at 2am if they are used to going to bed at 4am  — they often can delay their sleep if they try.  Thus it was proposed that they could normalize their sleep by going to bed later and later until they rolled around the clock to a normal sleep time.  If they started at 4am they would be told to go to sleep the next day at 7am, then 10am then 1pm and so on until they reached a normal bedtime.

Described this way, it’s easy to see that chronotherapy for DSPS consists of temporarily following a schedule like that of someone with N24.

This first phase of chronotherapy is supposed to be followed by a second stabilization phase once the desired sleep time is reached. In the stabilization phase the subject is supposed to rigidly stick to the new bedtime and wake time.

Sometimes this works.  Chronotherapy has been successful in some individuals.  But not always.  The N24 state, once entered into, is not so easy to reverse. In some persons, it is irreversible and they find that chronotherapy, far from curing their circadian problem, has instead converted it to a new, more impairing form.

There are two reasons why the transition to N24 can be difficult to reverse.

The first reason has to do with the relative phase of sleep compared to the phase of the body’s circadian rhythm which determines the phase response curve to light.  In many cases of DSPS the delay of the sleep cycle relative to the light PRC means that such “nite owls” are asleep during the time at which the body need to be exposed to light in order to advance the timing of the circadian rhythm.  When doing chronotherapy one goes to bed even later relative to the PRC. This decreases light exposure during the phase advance portion of the PRC and increases the light exposure during the phase delay portion of the PRC, causing a progressive delay of the circadian rhythm.  The circadian rhythm determines the rhythm of sleep propensity so that delays as well.  This sets up a positive feedback effect which tends to perpetuate the N24 state once it has been started.  To reverse N24 once this feedback loop is started is very difficult.

A second reason may relate to findings in studies of animals on non-24 hour schedules (produced by a non-24 hour zeitgeber such as lights that go on and off every 25 hours). It has been found that prolonged maintenance on such a schedule changes the apparent period of the circadian rhythm, so that even when released from the non-24 hour zeitgeber into an environment of constant light or dark they continue to show signs of their prior N24 schedule [3]. This was the reason cited in the original NEJM article.

How large is the risk of inducing N24 after chronotherapy?  The NEJM article mentions 3 patients, which seems small until one recalls that the original article on chronotherapy in 1981 only cited 5 successful cases [1]. There have been other reports of successful chronotherapy since then, but usually with small numbers of patients.  Published cases of chronotherapy leading to N24 have been fewer; but as I mentioned, I have heard personally from other people in whom this has happened.  There has been no systematic attempt to determine the relative risk.  But given the small numbers of reported chronotherapy successes, the even smaller numbers of conversion to N24 cannot be considered negligible.

It may be that this risk could be reduced by the additional use of light boxes and dark therapy during the stabilization phase of chronotherapy. But this is speculation.  The authors of the NEJM article suggest a slow advance of DSPS using light therapy as preferable to chronotherapy.

Of course not all DSPS patients will respond to slow phase advance by light therapy. For those who don’t the possibility of chronotherapy is tempting.

I am not someone who likes to make blanket statements. I would not suggest that chronotherapy be abandoned entirely. It does work for some.  Nor am I criticizing the researchers who invented chronotherapy.  Since prior to that there were no treatments at all for DSPS it was an important advance, and one that may still have its uses.  But what I would say is that anyone starting chronotherapy needs to know that there is a  risk it could make their circadian disorder worse. It is a calculated risk, although one in which we have little data to make that calculation accurately.

Many web sites and even medical texts mention the use of chronotherapy.  Very few mention the risk that it can induce N24.  One would have thought that an article in the prestigious New England Journal of Medicine would have been enough to get the word out, but clearly this aspect needs to be more widely discussed, which is why I am posting this.

—Posted by LivingWithN24 (James Fadden)

References:

1. Czeisler CA, Richardson GS, Coleman RM, Zimmerman JC, Moore-Ede MC, Dement WC, Weitzman ED. Chronotherapy: resetting the circadian clocks of patients with delayed sleep phase insomnia. Sleep. 1981;4(1):1-21.

2. Oren DA, Wehr TA. Hypernyctohemeral syndrome after chronotherapy for delayed sleep phase syndrome. N Engl J Med. 1992 Dec 10;327(24):1762.

3.Pittendrigh CS, Daan S. A functional analysis of circadian pacemakers in nocturnal rodents. 1. The stability and lability of spontaneous frequency. J Comp Physiol [AI 1976;106:223-52.

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