To stay up to date on the cutting-edge of health and performance, H.V.M.N. Research Lead Dr. Brianna Stubbs tends to read a lot of scientific literature...a lot. Every month, she will dive into the latest and most exciting research papers by walking us through the experiment process, dissecting the results and implications, and candidly sharing her own thoughts on the study and subject as a whole.

This month, Dr. Stubbs dives into three papers that studied the influence of the circadian rhythm has on sleep, blood pressure, and side effects of medical treatment.

1. A Length Polymorphism in the Circadian Clock Gene PER3 is Linked to Delayed Sleep Phase and Extreme Diurnal Preference
2. Genetic Variants Predict Optimal Timing of Radiotherapy to Reduce Side-effects in Breast Cancer Patients
3. Female C57BL/6J in Mice Lacking the Circadian Clock Protein PER1 are Protected from Non-dipping Hypertension

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Transcription

Overview

Today, we are going to be thinking about the tick tock of our biological clock.

This phrase might make you think about about aging or reproductive age; so clocks on something of a macro-scale over our lifespan. Actually, many processes inside our bodies are regulated on more of a micro-scale by our biological clock. Once thought of as being just inside our brain, countless genetically controlled clocks tick inside different parts of our bodies, such as the liver, kidneys, and heart. Among other things, they initiate many metabolic processes, ensuring that these occur at the optimal time of day. Internal processes that are triggered on a daily cycle are referred to as ‘circadian,’ and these are traditionally linked to the light/dark cycle.

Those who have flown overseas or from one North American coast to another will likely understand what it feels like when the body’s rhythms are out of sync. Traveling long distances across multiple time zones throws off the usual clock-setting cues, or zeitgebers, such as the daily light-dark cycle. Jet lag can cause a variety of temporary symptoms, including dizziness, irritability, and indigestion.

Longer-term perturbations of these rhythms can have lasting effects on the body. Researchers have also found that, in rodents, mutations in circadian clock genes can cause obesity, metabolic syndrome (a cluster of conditions that includes high sugar and low insulin levels in the blood), and diabetes. A number of epidemiological studies have shown that people who work night shifts are at a higher risk for these conditions as well.

The primary circadian clock in mammals is located in part of the brain called the suprachiasmatic nucleus (or nuclei) (SCN), a pair of distinct groups of cells located in the hypothalamus. Destruction of the SCN results in the complete absence of a regular sleep–wake rhythm. The SCN receives information about illumination through the eyes. The retina of the eye contains "classical" photoreceptors ("rods" and "cones"), which are used for conventional vision. But the retina also contains specialized ganglion cells that are directly photosensitive, and project directly to the SCN, where they help in the entrainment (synchronization) of this master circadian clock.

The SCN takes the information on the lengths of the day and night from the retina, interprets it, and passes it on to the pineal gland, a tiny structure shaped like a pine cone and located on the epithalamus. In response, the pineal secretes the hormone melatonin. Secretion of melatonin peaks at night and ebbs during the day and its presence provides information about night-length.

The classic phase markers for measuring the timing of a mammal's circadian rhythm are: melatonin secretion by the pineal gland, core body temperature minimum, and plasma level of cortisol.

However, as I mentioned earlier, more-or-less independent circadian rhythms are found in many organs and cells in the body outside the SCN, the "master clock". Indeed, neuroscientist Joseph Takahashi and colleagues stated in a 2013 article that "almost every cell in the body contains a circadian clock." Clock systems are a sort of core, primordial part of our genome that instruct and prepare cells for the work of using nutrients, moving around, breathing, and other fundamental processes.

The 2017 Nobel Prize in medicine was awarded to three scientists who discovered the clock genes that control the circadian rhythm, or the body’s natural day-night cycle. All three scientists made the discovery in the 1980s and based their work on fruit flies. Michael Rosbash and Jeffrey Hall collaborated to isolate the “period gene” that is key to regulating the circadian rhythm. This gene (called ‘per’) encodes for a protein that builds up at night and then degrades during the day; different levels of the protein affect how our body adapts to different times of day. Oscillations in levels of both per transcript and its corresponding protein have a period of approximately 24 hours and together play a central role in the molecular mechanism of the biological clock driving circadian rhythms. Mutations in the per gene can shorten, lengthen, and even abolish the period of the circadian rhythm. It even exists in plants!

Today, we will look at some classic research on gene variations in the PER gene and how that affects if you are a ‘lark’ or ‘owl’ and fast forward to research that has come out in the last few months that has shown that PER variants can even alter disease risk and response to medical treatments.

A Length Polymorphism in the Circadian Clock Gene PER3 is Linked to Delayed Sleep Phase and Extreme Diurnal Preference

Let’s start off with a 2003 paper that showed that variations in PER can affect preference to certain times of the day. In this study the authors were interested to see if differences in PER variant affected ‘diurnal preference,’ which basically means if you are a morning lark or a night owl. 484 healthy people completed a survey called the Horne-Östberg questionnaire. They also recruited 16 people with a relatively rare condition known as delayed sleep phase syndrome DSPS, which is a pathologic extremes of diurnal preference. The questionnaire was designed to see how strong a preference people had for time of day.

Subjects also gave saliva samples to allow for genotyping of their PER genes. They were looking at a polymorphism that changed the length of the PER gene by the number of times it was repeated, either 4 or 5 units. When discussing genetics, you need to know that we have two copies of each gene, one on each of our paired chromosomes; one of these is maternal and the other is paternal. You can either be homozygous for a gene, meaning you have the same variant in both of your copies, or heterozygous, meaning you have different variants on each copy of the gene. So in this study people could be homozygous for 4 repeat variant of PER, homozygous for 5 repeat variant of PER or heterozygous with one copy of the 4 repeat variant and one copy of the 5. From the healthy people, the researchers isolated the 7% of people with the most extreme morning preference, the 7% with the most extreme evening preference and a control group with an intermediate preference. They found a trend that the 5 repeat variant of the PER gene was significantly more common in people with strong morning preference but was far less common in those with an evening preference. In fact, when you looked at the people with delayed sleep phase syndrome (so pathological evening preference) there were NO people who were homozygous with two copies of the 5 repeat variant.

The authors suggest that having two copies of the 5 repeat variant of PER is linked to morning preference whilst having two copies of the 4 repeat would make you more likely to have an evening preference. This probably works because the repeated site is a place where the gene can be ‘phosphorlyated’ which alters its activity. If you have an extra repeat (5 vs 4) then you have more chance of this phosphorylation occurring. The authors say that understanding how PER repeat is linked to preference may help us to diagnose people with clinical diurnal preferences and also might provide a molecular target to help us to modulate the biological clock. But maybe in the shorter term, the ability to tolerate night shift work, time zone transitions, and artificial time cues in a 24-hour society is likely to depend upon the presence of specific clock gene variants, such as the one reported here, so as gene sequencing service become more common perhaps you will be able to give your boss a scientific reason for why you work best at 1am..

Genetic Variants Predict Optimal Timing of Radiotherapy to Reduce Side-effects in Breast Cancer Patients

So, we’ve seen that we might have a greater chance of being a lark or an owl based on our PER genes, but what if there’s more to it than that? What about if I told you your PER variant would predict side effects and response to medical treatments? This was explored in a study by genetic scientists at the University of Leicester. They show that radiotherapy toxicity - the side effects from radiotherapy - can be reduced by scheduling treatment according to the body’s circadian rhythm.

The study tested 1007 participants for two gene variants to decipher the nature of their circadian rhythms. One of the genes they were looking at was PER, the same gene as we discussed in the previous paper.

All participants had either previously undergone a course of radiotherapy or were currently on one. The treatment, which uses high-energy rays to target cancer cells, has typical side effects including skin pain, burning and swelling immediately after treatment. The issues can manifest in nerve damage and weaker bones later down the line. According to the researchers, around 90 percent of operable breast cancer patients are treated with radiotherapy, of which 45 percent experience nasty side effects in one way or another.

One key finding was that, no matter what the gene variant, breast cancer patients suffered worse side effects to radiotherapy in the morning. They found that 24 percent of patients treated in the morning had bright red skin after radiotherapy compared to 11 percent of those treated in the afternoon. Looking at peoples genes variants showed that a bias towards evening-ness with the 4 repeat PER variant made it more likely that you would get side effects if treated in the morning, with those who were 4 repeat homozygous experiencing increased toxicity if treated in the morning compared with the afternoon.

There are several potential physiological mechanisms to explain how time could affect reactions to irradiation, all linked to circadian changes that I mentioned earlier. The list includes melatonin, cortisol, inflammatory factors or cell proliferation/DNA damage. Melatonin has antioxidant properties, has been shown to be radioprotective in mice and reduce oral mucositis in irradiated rats. Cortisol levels can be used as a marker of stress and can affect inflammatory markers. Cortisol levels can influence the rate of cell division and may be associated with possible increased cellular division of skin in the morning compared with later in the day. That all said, this study wasn’t designed to really find out the mechanisms, this would need looking at in future work.

So, this study shows that getting radiotherapy in the afternoon is best for avoiding side effects, but if you are a morning person, you should try extra hard to get the afternoon appointment. When commenting on this story, clinicians pointed out that at the moment, people are scheduling appointments in around when they have to pick the kids up from school or they get given a slot whenever the machine is available. The researchers suggest that to implement their findings, all cancer patients should have their genes sequenced at the time of diagnosis to make sure that morning people get priority on afternoon treatment. It’s an upfront cost but it could save a lot of people from getting bad side effects. Those people will be admitted to hospital for their reaction so the money might be a good investment. There is still work to be done to confirm this in larger trials, and as I said to understand the mechanisms responsible, but clearly differences in our internal clock genes could have a future important role for personalized medicine.

Female C57BL/6J Mice Lacking the Circadian Clock Protein PER1 are Protected from Non-dipping Hypertension

Aside from differences in individuals, there may be differences on the sex level between the function of our circadian clocks.

Staying with the focus on the PER gene here, this paper published in the American Journal of Physiology, suggests that genetically knocking out PER1 acts differently in males versus females and may protect females from heart disease. The study is the first to analyse circadian blood pressure rhythms in female mice and distinguishes further biological differences between males and females. It’s an important subtlety when all types of science, from basic research through to sports science clinical studies commonly test interventions or study physiology in males to minimize the noise created by female hormonal fluctuations.

The body’s circadian clock, contributes to normal variations in blood pressure and heart function over the course of the day. For the majority of healthy humans, blood pressure dips at night, but individuals who do not experience this temporary drop, called ‘non-dippers’ are more likely to develop heart disease.

As I’ve said, the circadian clock has wide reaching impact, regulating close to half of all genes in the body, including those important for blood pressure regulation. Previous research has shown that male mice that are missing the clock gene (PER1) become non-dippers and have a higher risk for heart and kidney disease.

In this study, the research team studied the circadian response and blood pressure of female mice that lack PER1 and compared them with a healthy female control group. The researchers found that on both low- and high-salt diets, both groups showed to retain an apparent circadian rhythm of blood pressure. Unlike the male mice in previous research, the females without PER1 showed normal dips in blood pressure overnight. This illustrates that women may have a different response to clock gene activation, not being as reliant as males on PER1 to maintain their circadian changes in blood pressure. It isn’t clear exactly how this maps into humans (especially as we don’t knock out whole genes in people) however, the findings are consistent with research showing that premenopausal women are less likely to be non-dippers than men of the same age and, in general, females are protected compared with males with respect to cardiovascular morbidities.

This study essentially represents an important step in understanding biological differences between males and females in the regulation of cardiovascular function by the circadian clock gene. So not only is there much to be done to understand how these clock genes alter physiology, but we also need to take into account that responses could differ between men and women, so let’s not over-fit based on 50% of the population!

Conclusion

To me, there is something poetic and beautiful about the idea of our clock genes all in a state of constant flux together. It’s amazing to think about how dynamic our genes are. Really, how we think about our DNA has changed over the years: from thinking of it as a static line of code, to realizing that not only is DNA responsive to our environment (with things like exercise, or ketosis being able to impact gene expression), but it also has this inbuilt rhythm, like a heartbeat, that has huge implications for our life and health.

With the advent of consumer genetics, we might be able to accelerate the translation of our basic science understanding into meaningful observations about our own genetics: A few years ago, 23andMe researchers completed a genome wide association study that uncovered 15 genetic variants associated with being a morning person. Some of the variants were in newly associated genes, but others were in genes with well-established influence on circadian rhythms, such as PER2. That said, PER isn’t part of their standard report; but they can tell you at how variants in two genes— BTBD9 and ADA — may influence your ability to drift off into a deep sleep, and how likely you are to move about while you slumber. Hopefully our discussion today has given you something to sleep on, watch out for more discoveries that help us to optimize our lives and our health based on our clock genetics.

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