Hard Times for My Ancestors Have Marked My Genome

In this post, I present one small part of the tale of how the tribulations of my ancestors have left their mark upon my genome. I’m not sure who is reading this blog. I don’t know the average level of understanding of genetics among my readers. I have recently participated in the online discussions at 23andMe, where it is clear that many subscribers are struggling with the basics. This post will therefore contain a bit more background information than the average genomics blog. Some of the more technical information and citations are included in the footnotes.

Inherited genetic variation makes us different from each other. We all have the same genes, but every gene comes in different “flavors,” called alleles. Different alleles of a gene differ in their DNA sequence. Under some circumstances, alleles that eliminate the function of a gene can confer a selective advantage, meaning that people carrying that allele are likely to have more offspring. Changes in the frequency of a particular allele in a population (the “allele frequency”) over time can alert researchers to interesting problems in biology and medicine.

Most of the single nucleotide polymorphisms (SNPs) that are typed by 23andMe are “neutral.” They are sequence variants in parts of the genome that do not encode proteins. It doesn’t matter which base is present at that position, so natural selection does not change the frequency of a particular variant of this type directly. A small fraction of the SNPs typed by 23andMe are diagnostic for a variant allele of a gene that changes the sequence of the protein encoded by that gene. These variants have been discovered through research on people affected by an inherited disorder. A probe specific for the disease allele has been incorporated into the tests done by 23andMe. In my last post, I discussed three such variant alleles that I carry.

The variant allele that I carry for the PEX1 gene, PEX1-G843D, has an allele frequency of 0.001-0.002 (1). This means that if we look at all of the PEX1 alleles in a population, 0.1-0.2% of the alleles are the PEX1-G843D variant. The PEX1-G843D allele is a bad thing. While carriers like myself are unaffected, homozygotes (PEX1-G843D/PEX1-G843D) usually die before they reach one year of age. Why doesn’t natural selection “get rid of” this nasty allele?

The frequency of PEX1-G843D is at most 0.002. Assume for simplicity that there are no other variant alleles of PEX1, so the frequency of the wild-type (normal) allele is 0.998. If people mate at random without regard to their PEX1 genotype, we can calculate the frequency of the three possible genotypes as PEX1/PEX1 = 99.6%, PEX1/PEX1-G843D = 0.4%, and PEX1-G843D/PEX1-G843D = 0.0004% or 4/1,000,000 (2). PEX1-G843D is subject to negative selection, but this no longer changes the allele frequency of PEX1-G843D very much. There are about 1000 times as many heterozygotes (PEX1/PEX1-G843D) as there are homozygotes (PEX1-G843D/PEX1-G843D), so the allele frequency can’t be driven much lower by selection alone.

What about hemochromatosis? I am a “compound heterozygote” for two different variant alleles of HFE: HFE-H63D and HFE-C282Y. HFE-H63D has an allele frequency of 0.108 (10.8%) in a large diverse population sample from the Exome Sequencing Project and an allele frequency of 0.179 (17.9%) in a sample confined to Europeans (3). It is hardly surprising that I carry one HFE-H63D allele given my European ancestry. HFE-C282Y has an allele frequency of 0.047 in the sample from the Exome Sequencing Project and an allele frequency of 0.042 in a sample confined to Europeans (4). The protein encoded by the HFE-H63D allele has greatly reduced function, while the protein encoded by the HFE-C282Y allele is almost completely nonfunctional.

We know from studies of patients with hemochromatosis that the vast majority are HFE-C282Y/HFE-C282Y. A small fraction of hemochromatosis patients are HFE-C282Y/HFE-H63D like me. The rest have other variant alleles of HFE, or variant alleles of one of four other genes that predispose to hemochromatosis (5). The high frequency of variant HFE alelles raises an interesting question. Why might being a carrier for an inherited disorder be a good thing?

There are plenty of examples of disease genes that confer an advantage on carriers. Among people of European descent, the second most common inherited disorder (after hemochromatosis) is cystic fibrosis, resulting from defects in the CFTR gene. It is the most frequent inherited disorder leading to childhood deaths. The cumulative allele frequency for all variant alleles (there are many) is around 0.03 – 0.05 in people of European descent (6). Taking the low number (0.03) gives us the following genotype frequencies: CFTR/CFTR = 94%, CFTR/CFTR-variant = 5.8%, and CFTR-variant/CFTR-variant = 0.09% or 9/10,000 (7).

Almost 1/1000 children born to parents of European descent are afflicted with cystic fibrosis, in contrast to 4/1,000,000 for PEX1 variants (Zellweger Syndrome). About one person out of twenty of European descent is a carrier of a variant allele of CFTR, while only one person in 250 is a carrier of PEX1-G843D. People who are heterozygous for a variant allele of CFTR are healthy, but have additional genetic advantages: they are resistant to cholera and typhoid fever. Although these diseases are present outside of Europe, carriers of cystic fibrosis have salty sweat, and the loss of salt in hot climates may outweigh the advantages of disease resistance.

There are other examples of disease resistance conferred to carriers of genetic disorders. Three well-known inherited diseases confer resistance to malaria: sickle-cell anemia (HBB), thallasemia (HBB), and Favism or G6PD deficiency (G6PD). In a sample of 114 chromosomes from sub-Saharan Africa, the frequency of the sickle-cell allele of HBB was 11.4%, but it is not found in European populations. In some African populations, the allele frequency of a common variant of G6PD conferring malaria resistance is 20%. Other loss-of-function alleles of G6PD are common in Mediterranean or South Asian populations (8, 9).

Why might loss of function of the HFE gene, which leads to hemochromatosis, have a selective advantage? There are two interesting ideas about this. The first idea is that reduced function of HFE was a useful adaptation to the neolithic diet (10). When our ancestors switched from being hunter-gatherers to the practice of agriculture, the amount of red meat (a great source of iron) in people’s diets fell. The switch to a grain-based diet meant that careful biological regulation of the amount of iron taken in from the diet was no longer optimal. People with a defect in the signaling mechanism controlling iron uptake (of which the HFE gene product is a part) would experience iron overload, but might have an advantage during times of iron starvation, because it takes longer to deplete their body’s supply of iron.

While this allowed the allele frequency for variant alleles of HFE to rise, it might not fully account for the high frequency of HFE-H63D among people of European descent. There is a really great story here, first proposed by Sharon Moalem in a scientific paper (11) and popularized in his book Survival of the Sickest (12). Dr. Moalem has proposed that variant alleles of HFE rose to their current high frequencies because they confer resistance to bubonic plague, also known as the Black Death.

The Black Death is a bacterial infection caused by Yersinia pestis. People are infected with the bacterium when they are bitten by infected fleas, which are carried throughout the population by rats. When the plague bacterium enters the bloodstream, it is attacked by white blood cells called macrophages, which travel to the lymph nodes to carry out the destruction of the invaders. For most bacterial infections, this is usually a good strategy. However, the plague bacterium is often able to survive as a passenger in the macrophage, permitting the bacterium to attack the lymph nodes, causing one of the ghastly symptoms of bubonic plague: lymph nodes that swell to the size of an egg, sometimes bursting through the skin.

The plague bacterium needs nutrients to thrive, and one nutrient in particular is usually in short supply: iron. While people with reduced HFE function generally experience iron overload, this does not affect all cells in the body equally. Macrophages from people carrying variant alleles of HFE are deficient in iron. The iron-poor environment in the macrophage among HFE-deficient people allows the macrophage to gain the upper hand against the bacterium. The Black Death of the 14th century killed 30-50% of the population in a number of European countries. If variant HFE alleles conferred an advantage against this devastation, the allele frequency could have risen sharply among the survivors. While the Black Death is no longer part of the landscape in Europe, there is not much selection against variant HFE alleles. People with hemochromatosis do not usually display symptoms until they are past the age where most people have already had children. Because hemochromatosis does not interfere with reproduction, there is no selection against it.

In my case, it is possible that there was selection for incresed body stores of iron (and hence reduced HFE function) in my immediate ancestry. Both of my parents survived starvation during World War II. My mother, who is Dutch, lived in Holland through the entire war, and endured the Hongerwinter (“hunger winter”) of 1944. My father, who was Hungarian, spent the last 18 months of the war in a Soviet POW camp under harsh conditions. Both of my parents survived conditions in which there was widespread death from malnutrition, or from causes in which malnutrition was a factor.

I don’t wish to make light of my parent’s experience, or of the experience of my more remote ancestors who lived through the Black Death. Yet we have the genetic hand that we were dealt, and it is up to each of us to play it well. I intend to be evaluated for iron overload by a physician, and have already changed my diet, sharply reducing my intake of red meat. Humor is also an important aspect of health. In that spirit, I made this slide for one of my recent talks.

In my next post, I will go more deeply into my ancestry, as revealed by genetic testing.


Footnotes

1. The allele frequency of the PEX1-G843D allele is taken from the OMIM entry for PEX1

2. The frequency of the three PEX1 genotypes is calculated given an allele frequency of 0.002 for PEX1-G843D and 0.998 for the normal PEX1 allele as follows:
PEX1/PEX1 = 0.998 * 0.998 = 0.996 or 99.6%
PEX1/PEX1-G843D = 2 * 0.998 * 0.002 = 0.003992 = 0.4%
PEX1-G843D/PEX1-G843D = 0.002 * 0.002 = 0.000004 or 4/1,000,000

3. The allele frequency for HFE-H63D is taken from the dbSNP entry for rs1799945.

4. The allele frequency for HFE-C282Y is taken from the dbSNP entry for rs1800562.

5. Please see the OMIM entry for HFE and hemochromatosis  and for four other genes causing hemochromatosis (HJV, HAMP, TFR2, and SLC40A1).

6. Please see the OMIM entry for CFTR.

7. The frequency of the three CFTR genotypes is calculated given an allele frequency of 0.03 for all CFTR-variant alleles and 0.97 for the normal CFTR allele as follows:
CFTR/CFTR = 0.97 * 0.97 = 0.9409
CFTR/CFTR-variant = 2 * 0.97 * 0.03 = 0.0582
CFTR-variant/CFTR-variant = 0.03 * 0.03 = 0.0009 or 9/10,000

8. Please see the OMIM entry for HBB for more information about sickle-cell anemia and thalassemia. 

9. Please see the OMIM entry for G6PD for more information about favism.

10. Christopher Naugler (2008) Hemochromatosis: A Neolithic adaptation to cereal grain diets. Medical Hypotheses 70: 691-692.

11. S. Moalem, M.E. Percy, T.P.A. Kruck, and R.R. Gelbart (2002) Epidemic pathogenic selection: an explanation for hereditary hemochromatosis? Medical Hypotheses 59: 325-329.

12. Sharon Moalem with Jonathan Price (2008) Survival of the Sickest, Harper Perennial.

Getting My Genome Done

I am a geneticist with a varied career that has included research and teaching at a variety of academic institutions. In 2000, I shut down my research lab and took a job in bioinformatics, just as the human and mouse genome sequences were being completed. In November of 2011, I moved to a new position at the University of New Mexico, funded in part by the National Human Genome Research Institute. My new position involves teaching and public outreach. Recent progress in human genomics has been spectacular, and it is a great story to tell. I thought that knowledge about my own genome would motivate my learning about human genetics and would also personalize my presentations, so I decided to “get my genome done.”

There are several ways of getting a look at your own genome. Of the direct-to-consumer companies, I liked 23andMe. At the 2011 SACNAS National Conference in October, I heard a talk by Dr. Joanna Mountain, Senior Director of Research at 23andMe. Dr. Mountain talked us through 23andMe’s website as seen by a user. The 23andMe website offers information on inherited health conditions and ancestry based on a person’s genome. I liked their user interface. They present results in language accessible to people without an extensive background in science. Users are only a few clicks away from full technical data, including complete raw data that can be uploaded to third-party sites for further analysis.

I signed up for 23andMe using their website, and soon received a kit in the mail for sample collection. They recover DNA from saliva using a very clever method. Following the illustrated instructions, I spit into a plastic tube equipped with a funnel. When my saliva reached the fill line, I flipped a cap into place that dumped a solution into the saliva sample. I capped the tube and inverted it a few times. As I did this, I saw the familiar sight of DNA coming out of solution in an ethanol precipitation. I have isolated plenty of DNA in my days as a researcher, but this was the first time that it was my own. I packed the tube in the postpaid return mailer, dropped it off at the Post Office, and waited.

After a few weeks, I got an email from 23andMe that my results were ready. I had purchased their only offering at the time, a survey of my genotype using the Illumina OmniExpress Plus Genotyping BeadChip. This technology allows genotyping of a human DNA sample at about one million genomic sites. The sites that are genotyped are Single Nucleotide Polymorphisms (SNPs) that have been identified as sites of variation in survey sequencing of human populations. The 23andMe chip includes some SNPs that are the sites of mutation in well-studied genetic disorders. For example, the chip tests for 31 different sequence variants of the CFTR gene associated with Cystic Fibrosis.

Although I am healthy and free from any known genetic disease, I looked at my Carrier Status. The screenshot below shows part of the 23andMe report.

There are 44 genetic disorders listed on this page. The disorders are listed in alphabetical order. If you have a variant allele for any of them, it sorts to the top of the page. I had two: Zellweger Syndrome Spectrum, and variants associated with hemochromatosis, a disorder in which excess iron is taken in from the diet.

The Zellweger Syndrome Spectrum gene tested for is PEX1, a gene required for the normal formation of peroxisomes. Peroxisomes are membrane-bound vesicles inside of cells that are required for the catabolism of fatty acids and other compounds. Fortunately for me, I am a carrier, which means that I am heterozygous. I have one working copy of PEX1 and one bad copy. There are no health consequences for carriers. People homozygous for the allele of PEX1 that I carry generally die before they are one year old. This is why this gene is listed on the Carrier Status page; no one homozygous for the mutant PEX1 allele G843D has a computer, a credit card, and a 23andMe account.

My Hemochromatosis report is more complex. I have two different variant alleles of the HFE gene, one of which slightly predisposes to hemochromatosis, while the other causes a considerable increase in risk of the disease. Here is a screenshot of the page that appears when you click on the Hemochromatosis link.

There is a link to a technical report. The technical report is very detailed. Part of it is shown below.

The good news is that my risk for developing hemochromatosis is quite low. Nevertheless, I decided to modify my diet and to ask for some specialized tests the next time I visit a doctor. I will discuss this in greater detail in another post.

There are also discussion forums at 23andMe. I participated in these for a couple of weeks before launching this blog. Not everyone has training in genetics, even among 23andMe subscribers, so I will take this opportunity to explain the language used in the technical report.

Most genes encode proteins. A protein (polypeptide) is a chain of amino acids; there are twenty primary amino acids that make up the set that can be encoded by the 64 three-base codons of the genetic code. There are single letter codes for each of the twenty primary amino acids. The HFE gene encodes a protein 348 amino acids long. The H63D allele changes the 63rd amino acid from histidine (H) to aspartic acid (D). The C282Y allele changes the 282nd amino acid from cysteine (C) to tyrosine (Y).

The C282Y allele results in a significant loss of function of the HFE protein. The cysteine residue at that position is highly conserved, meaning that when you look at the HFE gene in other organisms, there is usually a cysteine at that position. This is a highly significant risk allele. From the OMIM entry:

“In patients with hemochromatosis, Feder et al. (1996) identified an 845G-A transition in the HFE gene (which they referred to as HLA-H or ‘cDNA 24′), resulting in a cys282-to-tyr (C282Y) substitution. This missense mutation occurs in a highly conserved residue involved in the intramolecular disulfide bridging of MHC class I proteins, and could therefore disrupt the structure and function of this protein. Using an allele-specific oligonucleotide-ligation assay on their group of 178 patients, they detected the C282Y mutation in 85% of all HFE chromosomes. In contrast, only 10 of the 310 control chromosomes (3.2%) carried the mutation, a carrier frequency of 10/155 = 6.4%. One hundred forty-eight of 178 HH patients were homozygous for this mutation, 9 were heterozygous, and 21 carried only the normal allele. These numbers were extremely discrepant from Hardy-Weinberg equilibrium. The findings corroborated heterogeneity among the hemochromatosis patients, with 83% of cases related to C282Y homozygosity.”

In other words, looking at this from the perspective of a physician, most people who receive a clinical diagnosis of hemochromatosis are homozygous for the C282Y allele of HFE.

In contrast, also from the OMIM entry, the H63D allele of HFE confers a minor risk of hemochromatosis. Here is one part of the OMIM entry:

“Jouanolle et al. (1996) commented on the significance of the C282Y mutation on the basis of a group of 65 unrelated affected individuals who had been under study in France for more than 10 years and identified by stringent criteria. Homozygosity for the C282Y mutation was found in 59 of 65 patients (90.8%); 3 of the patients were compound heterozygotes for the C282Y mutation and the H63D mutation (613609.0002); 1 was homozygous for the H63D mutation; and 2 were heterozygous for H63D. These results corresponded to an allelic frequency of 93.1% for the C282Y and 5.4% for the H63D mutations, respectively. Of note, the C282Y mutation was never observed in the family-based controls, whereas it was present in 5.8% of the general Breton population. This corresponds to a theoretical frequency of about 1 per 1,000 for the disease, which is slightly lower than generally estimated. In contrast, the H63D allelic frequency was nearly the same in both control groups (15% and 16.5% in the family-based and general population controls, respectively). While the experience of Jouanolle et al. (1996) appeared to indicate a close relationship of C282Y to hemochromatosis, the implication of the H63D variant was not clear.”

So, while the H63D allele of HFE appears to alter the function of HFE, it is almost as frequent among patients lacking a diagnosis of hemochromatosis as among those who are diagnosed with hemochromatosis. People have two alleles, so “having” the H63D allele in this case usually means also having a normal allele. I should also point out that there are other genes, different from HFE, that predispose to hemochromoatosis.

The Zellweger Syndrome Spectrum (PEX1) allele that I carry occurs at a frequency of around 0.2%. For hemochromatosis, among the 4,552 chromosomes sampled from the publicly-funded Exome Sequencing Project, the HFE-H63D allele occurs at a frequency of about 10.8%, while the HFE-C282Y allele occurs at a frequency of about 0.2%. Why is there such a wide range in the frequency of disease-causing alleles? I will cover that in my next post.