The genetics of the calico cat

In an earlier post, I detailed the life and work of Nettie Maria Stevens, the namesake of our new calico cat. Nettie Stevens is known for her discovery of the X and Y chromosomes as the basis for sex determination. While researching the post, I found myself in another internet rabbit hole and I wanted to share some of the things I learned about cat genetics because, it turns out, calico cats are an excellent lesson in genetics.

The genetics of cat fur color (like the genetics of human hair color) are rather complex. There are many different genes that create the spectrum of coat colors and patterns found in domestic cats. In other mammals, white fur color alone has been linked to at least six genes (MITF, EDN3, EDNRB, PAX3, SOX10, and SNAI2). For this discussion, I will focus only on the gene loci that create the calico pattern: agouti (A), orange (O), and spotted (S). Based on the combined expression of these genes, some cats are tortoiseshell, a mix of black and orange and some cats are calico, a mix of black, orange, and white. Because the Orange gene is on the X chromosome, most torties and calico cats are female. There are rare cases of male cats with Klinefelter syndrome (XXY) that can have these fur color variants.

Figure 1 Schmidt-Kuntzel, et al., 2009

Agouti (gene name ASIP) encodes a protein that inactivates the melanocortin receptor and thus controls the distribution and amount of pigment cells (melanocytes) in the hair. The Orange gene has not yet been identified, but its position on the X chromosome has been mapped. Interestingly, the MC1R gene, which has been linked to red hair in humans and other mammals, is not involved in the coloring of calico cats because it isn't on the X chromosome. A recent study of the Syrian hamster, which shows sex-linked inheritance of yellow fur color, described the sex-linked yellow gene (Sly) as being independent of MC1R function and not likely homologous to the Orange gene in cats. However, recessive versions of the MC1R allele create orange/amber fur color in Norwegian forest cats. The gene for white spotting (S) has been linked to the KIT locus; more recent publications have shown that a retroviral insertion in the KIT oncogene caused white spotting, with a full insertion leading to the recessive all white mutation.

The genetics of the tortoiseshell/calico cat

If fur color followed the rules of Mendelian genetics, you would expect cats to be only black and white or orange and white, not two or three colors at once. The patterns observed in calico and tortoiseshell cats can be explained by a phenomenon called X chromosome inactivation. Somatic chromosomes come in even pairs, but the sex chromosomes are not created equal. The Y chromosome is rather small and does not encode many genes. In contrast, the X chromosome contains lots of protein-encoding genes. If an XX individual expressed all the genes on both X chromosomes, they would have a huge imbalance in protein expression in comparison with someone with XY. Organisms have different ways to level the compensate for these differences. In mammals, this involves silencing one of the X chromosomes in each cell with the XX genotype. In organisms like the fruit fly, the XY males just double the expression of the genes on the X chromosome.

The specifics of how this happens is actually pretty amazing. (For further details of the process, check out my post on Nessa Carey's book Junk DNA.) In short, a long non-coding RNA known as Xist (X-inactive specific transcript) turns off one copy of the X chromosome in each cell; on the opposite strand of DNA, a gene called Tsix is found. Xist and Tsix have mutually exclusive expression, which ensures that only one X chromosome is inactivated in each cell. X chromosome inactivation occurs at the 8 cell stage of the embryo. Different cells inactivate different copies of the X chromosome, which means some cells express alleles for orange and white and other cells express black and white alleles. These 8 cells then divide and produce the millions of cells that make up a cat. This random pattern of gene inactivation leads to the pattern observed in Nettie and other calicos.

Carbon copy (left) is a clone of Rainbow (right)

Importantly, because these traits are not strictly inherited by classic Mendelian patterns, it makes cloning cats a bit trickier than other animals. As highlighted in the book Frankenstein's Cat, scientists were disappointed when the cloned cat Carbon Copy ended up looking quite different from its clone mother. Had they thought more carefully about the genetics of calico cats, they might have picked a different breed for their experiment!

maneki-neko calico cat figurines are
thought to bring good luck

Calico cats like our Nettie offer excellent lessons in genetics as they higlight X-linked genes and dosage compensation. After all this reading, I think I will take advantage of cat genome sequencing to discover the specific mutations in our cat. I learned more than expected about cat genetics and hope to share these lessons with you soon. For now, if you want to learn more about the genetics of cats, you should check out Herding Hemingway's Cats by Kat Arney.

Ten Great Science Articles and Blog Posts From Across the Internet

Some websites do a weekly best of the web and, frankly, I wish I were that well read or that ambitious. In lieu of such lofty ambitions, I will share a listicle of some of my favorite science articles and blog posts. Enjoy! 

The Man with the Golden Blood: This Mosaic Science piece explores the market for rare blood; easily one of the best long reads I have found. This makes a good compliment to the Radiolab episode on Blood.

Brazil's Cancer Curse: Title aside, this is a great story by Sue Armstrong, whose book about p53 I recently reviewed. She digs deep into a Brazilian cancer cluster and how the doctors made the link to p53 mutations. The best part is the speculations about where this mutation arose and how it fixed in the population.

He Thinks He's Untouchable: Yes, this is BuzzFeed and yes, it is worthy of this list. This is one of many well researched and frankly exasperating stories about sexual harassment in academia. I have read too many of these stories, this one is definitely the most extreme of them all.

The Unique Merger That Made Ewe, You and You: A list of great science reporting would not be complete without something from Ed Yong. Frankly, it's difficult to choose just one. This one is a favorite simply because it addresses a subject dear to my heart: endosymbiotic evolution. 

Lessons of Immortality and Mortality From My Father, Carl Sagan: A bit far adrift for science articles, but this story from Carl Sagan's daughter was published at the perfect time in my parenting life–after the death of a close friend of the family. It helped me find a way to frame life and death in a way consistent with my perspective as a scientist and a way that was understandable to a young child.

Medical Research: Cell Division: A title that doesn't really capture the awesomeness at the heart of this story. In the vein of Rebecca Skloot's HeLa cell book, this story captures the ethical issues surrounding the Leonard Hayflick's creation of the WI-38 cell line from an aborted fetus. The author, Meredith Wadman, has a book related to this topic coming out in early 2017.

He may have invented one of neuroscience’s biggest advances. But you’ve never heard of him: Another title I hate hiding a story I love from STAT news. This one surrounds the invention of optogenetics in neuroscience. For me, it highlights the need for researchers to be sure readers and editors understand the implications of their work.
 
How Elizabeth Holmes's House of Cards Came Tumbling Down: Again, this story is not in my usual wheelhouse, but I have been following the story of the Theranos company with great fascination. This Vanity Fair piece gets into how Holmes was able to convince everyone around her that this idea was the next Unicorn and that it was scientifically sound, even though she neglected to get the scientists and clinicians on board from the beginning.

Who was Phineas Gage?:This piece from Sam Kean gives you a great sense of what his book The Dueling Neurosurgeons is about. This may be one of the weirdest stories in science, so it makes sense that it was likely the start of Kean's book about the brain.

How Your Cat is Making you Crazy:  I should be embarrassed to admit how many times I have shared this story from 2012, but I find it oddly compelling read about the strange effects of Toxoplasmosis (that stuff in cat poo) on the brains of humans and mice. It has all the things necessary for a good science article: cats, weird scientists, and unexpected results.

Junk DNA: Nessa Carey's new book about the actually important stuff in the genome.

In 2001, when the first draft of the human genome was completed, researchers were surprised to learn that only 2% of the human genome codes for proteins. At the time, scientists were very focused on proteins and thought that there would be a much larger number of protein-coding genes in the human genome due to our complexity. The term "junk DNA" has been used to describe the other 98% of the genome. With only 20,000 protein-coding genes, the human genome contains almost the same number of genes as the simple roundworm and model organism C. elegans. However, C. elegans has very little excess DNA, suggesting that this junk DNA could be part of the explanation for the increased complexity of humans. This is the starting point for Nessa Carey's second book Junk DNA: A Journey Through The Dark Matter of the Genome, which explains the importance of the non-coding portion of the genome.

Some scientists have argued that the term junk DNA should be scrapped for a more neutral term like non-coding DNA. They suggest that the term is dated and inaccurate. In addition, calling it junk is rather pejorative and is based on the protein-focused view of the genome. Carey's book nicely demonstrates that the other 98% isn't always junk.

What is the other 98% of the genome good for then? Some non-coding DNA has well-established functions. For example, the centromeres are the stretches of DNA that allow the chromosomes to attach to the cell's chromosome segregation apparatus (the mitotic spindle) when the cell copies and divides its DNA. Another example is the telomeres, the lengthy repeat regions of DNA at the ends of the chromosomes. Because telomeres shorten with every cell division, they are linked with aging.

Junk DNA also encodes several special types of RNAs, including long non-coding RNA (lncRNA), microRNA (miRNA), and small interfering RNA (siRNA), that control gene expression. One of the earliest described examples of these special RNAs is found in the biology of sex determination. In XX females, one X chromosome is inactivated to ensure that genes on the X chromosome are not overexpressed. This process, called X chromosome inactivation, is controlled by a gene called Xist (X-inactive specific transcript). Xist encodes a long non-coding RNA, which covers one X chromosome and inactivates it (Xi). Interestingly, on the opposite strand from Xist is a gene called Tsix, which is expressed on the active X chromosome (Xa). The expression of these genes is mutually exclusive, ensuring that only one X chromosome is activated. The Xist/Tsix story highlights the power of special RNAs in controlling gene expression. These RNAs are the subject of intense research in both basic and clinical settings. Carey describes several approved drugs and promising clinical trials based on anti-sense approaches.

In short, Junk DNA was quite readable and should be informative for readers at any level of knowledge about molecular biology. My only complaint about the book was Carey's decision not to include protein or gene names in her writing. In the first chapter, she explains that this was due to the fact that half of her readers find it disruptive. Instead, where applicable, she includes footnotes with the gene or protein names. Unfortunately for me, I am in the half that finds it disruptive to read footnotes to learn the name of the gene in question. Otherwise, the book was very up to date and comprehensive. I also liked her use of simple graphics to explain complex concepts in molecular biology. I recommend Junk DNA for those who want to learn more about why the non-coding regions of our DNA are not junk.

The genetic wonders of red hair

I recently finished reading Armand Leroi's Mutants, where I learned about some of the polymorphisms (small changes in DNA sequence) linked to variations in human skin and hair color, particularly red hair. With two different types of redheads in the house, I have always been curious about the genetic basis of this relatively rare trait.

red-haired mouse (from Flickr)

Hair and skin color are determined by the relative proportions of the two types of melanin pigment: eumelanin (dark brown) and pheomelanin (red/yellow). Large amounts eumelanin result in darker hair and very little produces blond hair. People with more pheomelanin have red hair. Red hair phenotypes can range from pale red to bright red or reddish brown, which is due to a balance of the two melanin types. Polymorphisms in the melanocortin 1 receptor gene (MC1R) are associated with variations in hair and skin color in mammals. The MC1R protein is a membrane receptor found only in melanin-producing cells (melanocytes). In response to melanocyte-stimulating hormones (MSHs), MC1R initiates a cascade of cellular events that turns on the production of pigment synthesizing genes, including pheomelanin or eumelanin. Essentially, MC1R determines pigmentation by regulating the relative proportion of eumelanin and pheomelanin.

The ancestral form of the MC1R allele produces eumelanin; variant alleles are less functional, decreasing eumelanin production or increasing the amount of yellow/red melanin. Variations in MC1R contribute to a spectrum of phenotypes, including freckling, red hair color, and sun sensitivity. MC1R alleles that disrupt function are present in ~80% of individuals with red hair and ~20% with brown-black hair. More than 80 MC1R allele variations have been identified in European populations. Association studies have shown that certain MC1R variants (p.D84E, p.R151C, p.R160W, p.D294H, p.R142H, and p.I155T) are linked with the red hair color phenotype.

One unanswered question is why these polymorphisms are more prevalent in Eurasians than in Africans. The current hypothesis is that paler skin permits better synthesis of Vitamin D in environments with less sunlight. A recent study using data from the 1000 Genomes Project revealed that the polymorphisms are also more common in Northern Europe than Southern Europe, which supports the Vitamin D hypothesis. Interestingly, a MC1R variant has been observed in Neanderthals, indicating that red hair and pale skin were also present in this population. A recent paper has shown that the Neanderthal MC1R variant is rare in Europeans, but can be found in East Asian populations. This result suggests that mutation of MC1R was a common mechanism to adapt to changes in sunlight intensity.

from David Fisher's lab

Some MC1R variants are associated with an increased risk of melanoma. Due to increased sun sensitivity and freckling of people with pale skin, this relationship seems obvious. However, darkly pigmented Caucasians with certain MC1R variants also show an increased incidence of melanoma. This result suggests that the MC1R pathway may have another role in the development of melanoma beyond differences in the ability to filter UV rays in light and dark skin. In fact, scientists think that MC1R may play a pigment-dependent and a pigment-independent role in skin carcinogenesis. There are a few hypotheses to explain the link between MC1R and melanoma. First, after UV exposure, cells with more pheomelanin show increases in DNA damage, which is correlated with increases in abnormal cell growth and proliferation, a hallmark of cancer. Second, a recent paper in Molecular Cell suggested that UV light triggers the interaction of a tumor suppressor called PTEN with MC1R. The tumor suppressor can interact with wild type MC1R, but not the red hair alleles of MC1R. The PTEN-MC1R interaction protects PTEN from degradation, which suppresses an oncogenic signaling pathway (PI3K/Akt). In contrast, MC1R variants do not interact with PTEN, allowing increased levels of oncogenic signaling pathways after UV irradiation. Unfortunately for redheads, sun exposure alone is not the sole mechanism for skin cancer. A Nature paper from David Fisher’s lab used a red-head mouse model with inactive MC1R to investigate a possible UV-independent pathway. They found that in the presence of the most common melanoma oncoprotein (BRAF 600E), ginger mice developed melanoma without UV exposure, while MC1R wild-type mice did not. Thus, shielding easily freckled skin from the sun may not be enough to protect from skin cancer for people with certain MC1R variants.

The association between red hair and melanoma suggested that there may be a scientific basis for the headline from 2014 that climate change was putting the red hair gene in danger of extinction. The story was exposed as alarmist and scientifically inaccurate. One of the many problems with the article is that they label the red hair gene as recessive. Because hair color is a complex phenotype, it is not surprising that the genetics of red hair are also complex. Red hair is usually inherited in a recessive manner, but it can also be dominant. A 2000 study showed that the inheritance pattern depends on the MC1R allele that is present: most alleles are recessive, but some alleles can be dominant. Individuals that are heterozygous for a mutant MC1R allele show variations in red hair color, beard color, or freckling. Thus, there is a dosage effect of MC1R variants on hair and skin color, which explains why some men have red beards and brown hair. These results also explain how two red-haired parents can (on rare occasions) have children that don't have red hair. Thankfully, the many variations in MC1R and the complex inheritance patterns mean that red hair isn't likely to die out any time soon.

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For more information, I recommend the following links:

Red hair color: the myth

Causes, origins, and history of red hair

Red heads feel pain differently A blog post from 23andme about the population genetics of red hair.

Armand Leroi's Mutants: where the Mutter Museum meets Geek Love

Mutants: On Genetic Variety and the Human Body by Armand Marie Leroi explores the genetic and developmental reasons for variations in the human form. I thought this book would be a good follow up from Inheritance, which focuses on rare genetic disorders that often have a relatively minor physical abnormality, such as eyes that are widely space (hypertelorism). Based on the cover and title, I expected the book to be a bit of a carnivalesque spectacle– part Mutter Museum, part Geek Love. In fact, the text treated these "mutants" respectfully and with due scientific diligence. The focus was generally on the developmental pathways that were altered in the mutations observed, but there was some exploration of the toll these mutations took on their bearer.

The book begins with the historical observations of mutants and the explanations for why these mutations arose. In the beginning, "monsters" were thought to be caused by the wrath of God. Eventually, scientists began to understand the developmental causes for these variations. In a chapter called "A Perfect Join", Leroi writes about conjoined twins, specifically how they form. Interestingly, conjoined twins are more likely to have a condition called situs inversus, where the internal organs are inverted on the left-right axis. In fact, the rare singleton birth with this condition has been useful in teaching us why our organs are oriented the way they are. Situs inversus is one symptom of Kartagener's syndrome, which is caused by a mutation in one of the proteins (dynein's outer arm) that control the movement of cilia (tiny projections on the surface of many different cell types). The other symptoms are chronic bronchitis (cilia are important for clearing the lung and nasal passages) and sterility in males (the same proteins are required for the movement of sperm). I always find it amazing how a small mutation in one protein can have such profound consequences. My favorite lines from the book describe how scientists study gene function: "It is actually quite hard to prove that a gene...does what one supposes. One way...is to eliminate the gene and watch what happens. This is rather like removing a car part...to see why it's there. Sometimes only a rear-view mirror falls off, but sometimes the car dies."

A funny story arises in the explanation of the Hox genes, which essentially control the pattern of embryonic development such that Hox1 makes a head, Hox2 makes a torso, etc. Mutations in Hox genes can lead to abnormal body plans, even changes in the number of vertebrae and ribs. About one in every ten adults has an extra pair of ribs, an abnormality that was first observed in a woman, leading the doctors on the case to conclude that it was evidence of the truth of Adam and Eve. In reality, the physiological variation occurs at equal rates in men and women.

The chapter on skin color explores the mutations that lead to albinism, piebaldism, and even red hair. Dozens of different mutations in MC1R (melanocortin 1 receptor), a protein that helps control pigmentation, have been described in red heads. (Because my house has three different types of red heads, I really fell into an Internet rabbit hole on this subject. I think I will write something more about this later.) The genetics of skin color are also complex and have been difficult to study due to the complicated social issues surrounding the subject. Leroi tells the story of a white woman living in Apartheid-era South Africa. Following a diagnosis with Cushing's disease, her adrenal glands were removed, which led to hyperpigmentation due to an abundance of the hormone melanotrophin. As her skin darkened, her social status and living conditions quickly changed. To me, the story highlights the problem with thinking that we can biologically define race. (A Troublesome Inheritance was released recently and covers this very topic, so watch this space for my review.)

Leroi also discusses the genetic basis for aging; in the simplest terms, aging is due to the "inability of natural selection to act against the mutations that cause disease in the very old." This hypothesis explains why dominant mutations like Huntington's can fix in the population. An experiment in fruit flies explored what would happen if only aged flies were reproducing; the effect should be an increase in genetic changes that promote longevity and fertility at an advanced age. After ten generations, the longevity of these flies increased by 30%; after fifty generations, life expectancy doubled. The flies were generally hardier, but the increased life expectancy came at a cost: the fruit flies were less active in their youth as they needed to preserve themselves to ensure survival and mating. This experiment supports the idea that aging comes at the expense of vitality in youth.

Overall, Mutants was quite readable; it had a nice balance of the science behind the mutations and the descriptions of the lives of the people who were affected with these mutations. I should note that the book was released in 2003, so some of the science is a bit out of date. For me, a good read is one that teaches me new science and spurs me to read and write more. Mutants definitely fits the bill in that regard. 

Inheritance by Sharon Moalem: an excellent new perspective on human genetics and epigenetics

Inheritance: How Our Genes Change Our Lives
– and Our Lives Change Our Genes, by Sharon Moalem, offers a unique perspective on the subject of human genetics. Moalem is a physician-scientist who specializes in rare diseases; his specialty has given him a keen eye for discerning subtle physical differences (phenotype) that are frequently linked to genetic differences (genotype). In some cases, the differences are insignificant. For example, if you have an extra row of eyelashes, you share something with Elizabeth Taylor, specifically a mutation in a gene called FOXC2. Other differences can serve as a diagnostic for rare genetic conditions. In one chapter, Moalem describes being at a dinner party when he noticed several physical traits that suggested that his hostess may have Noonan syndrome, which can be associated with heart disease and blood clotting. Another example is orbital hypertelorism, where the space between the eyes is large enough to accommodate another eye. This trait can be associated with Fanconi anemia, a blood disorder linked with an increased incidence of cancer. Wide-set eyes are commonly found in actresses and models (famous examples include Jackie Onassis and Michelle Pfeiffer). Our preference for certain physical traits may be explained by our interest in ensuring a good developmental and genetic history in our mates; Moalem suggests that facial features are an obvious indicator that brain and body development occurred properly.

Inheritance also highlights how minor differences in the sequence of our DNA can cause major differences in phenotypes. For example, congenital polycythemia (PFCP) is a genetic condition caused by a mutation in the EPOR gene that results in a greater number of red blood cells. This mutation gave Finnish athlete Eero Mäntyranta a distinct advantage in aerobic competition because his blood can carry more oxygen (essentially it's like he always has doped blood). However, PFCP can also lead to an increased risk of stroke. Thus, evolution took a different approach to solving the low oxygen problem for Sherpas: a mutation in the EPAS1 gene causes lower production of red blood cells, but increases the efficiency of oxygen delivery.  Interestingly, this mutation fixed in the population relatively quickly: Sherpas moved into their current low altitude environment around 1500. (This is a really fascinating story; if you want to read more, I recommend this Ed Yong piece.) These examples also underscore that while humans are ~99% similar, there is still a lot of variability in DNA sequence. In fact, in the 14 years since the first human genome was sequenced, we have learned that there really is no average genome. As I highlighted in my previous post on genomics, large-scale genomics projects (e.g., The 1000 Genomes Project) aim to get samples from a highly diverse set of people to remove the background noise so that significant differences can be identified.

Moalem also has an informative discussion of epigenetics (i.e., changes to DNA that do not occur at the sequence level). A great example in the book is the queen bee. Every bee in a colony is completely identical in their DNA sequence. How then does a queen bee become so different in size and function? Larval queens are fed royal jelly, which changes their DNA to allow them to express queen-specific genes. The protein DNA methyltransferase (Dnmt3) can methylate DNA and change its expression. In fact, if you shut down Dnmt3 in bee larvae, all of them become queen bees. The field of epigenetics is relatively young. However, there are already many fascinating ways in which our daily activities can alter the expression of our genes. The epigenome is dynamic and can be impacted by the things we eat and drink, the exercise we do, and, most surprisingly, the experiences we have. Accumulating evidence suggests that stress can alter your genome; in some cases, these changes can be inherited. The Radiolab episode Inheritance highlights a Swedish study that suggests that the eating habits of your grandfather could have an effect on your longevity. Geneticists are just beginning to find ways to track these changes and, more importantly, how to reprogram the methylated genes. As you can imagine, methylation is a very useful way for a cell to alter its gene expression. However, methylation can also mean the difference between a benign growth and a malignancy, making it an attractive target for therapeutic development.

Moalem makes an excellent argument for the support of the study of rare diseases. Most funding bodies think that funding diseases where many people are affected (e.g., cardiovascular disease, Alzheimer's) is the most cost-effective approach. However, studying the mutations and physiology of people with rare diseases can often shed light on the basic underpinnings of how normal cells work. For example, the study of familial hypercholesterolemia (FH) gave us insights into the function of LDL and HDL ("bad" and "good" cholesterol); these results facilitated the development of Lipitor, which has helped many people with elevated levels of LDL who do not have FH.

In short, Inheritance is immensely readable. The author uses clever and pertinent analogies. For example, he compares our cells to the production style of Toyota and Apple in that they stock only the supplies they need to avoid waste (an approach called production leveling). Another great example is when he is explaining a genetic condition that turns muscle into bone; he writes, "Osteoclasts are the Wreck-it Ralphs of the skeletal system. Osteoblasts are the Fix-It Felixes." Overall, the book is very topical and up to date; it would be an informative read for those who are well versed in genetics as well as those who are just becoming interested in the subject. Inheritance is definitely one of the best books I have read on the subject (a close second to Sam Kean's The Violinist's Thumb) and will be added to my list of great science reads.