Antagonistic Pleiotropy As a Widespread Mechanism for the Maintenance of Polymorphic Disease Alleles Ashley JR Carter* and Andrew Q Nguyen

Carter and Nguyen BMC Medical Genetics 2011, 12:160 http://www.biomedcentral.com/1471-2350/12/160

RESEARCHARTICLE Open Access Antagonistic pleiotropy as a widespread mechanism for the maintenance of polymorphic disease alleles Ashley JR Carter* and Andrew Q Nguyen

Abstract Background: Many serious diseases have a genetic basis which, from an evolutionary point of view, should have been selected against, resulting in very low frequencies. The remarkable sustained prevalence of a number of disease-associated alleles is therefore surprising. We believe that antagonistic pleiotropy, when multiple effects of a gene have opposing effects on fitness (e.g., sickle cell disease), may be more widespread than typically considered. We hypothesize that, rather than being an exception to the rule of genetic disorders, antagonistic pleiotropy may be common. Methods: We surveyed the medical literature in order to determine whether sufficient evidence exists to reassess the nature of antagonistic pleiotropy; from being considered an unusual scenario to one that is anticipated. We also used a simple population genetic model to examine the feasibility of antagonistic pleiotropy to act as a mechanism to maintain polymorphism for serious genetic disorders even if the benefits are subtle. Results: We identified a number of examples of antagonistic pleiotropy where the deleterious effect, the beneficial effect, and the exact molecular cause have been demonstrated. We also identified putative cases in which there is circumstantial evidence or a strong reason to expect antagonistic pleiotropy in a genetic disorder. The population genetic model demonstrates that alleles with severe deleterious health effects can be maintained at medically relevant frequencies with only minor beneficial pleiotropic effects. Conclusion: We believe that our identification of several cases of antagonistic pleiotropy, despite the lack of research on this question and the varied natures of the types of these disorders, speaks to both the underappreciated nature of this phenomenon and its potentially fundamental importance. If antagonistic pleiotropy is as common as our research suggests, this may explain why so many serious diseases, even apparently environmentally caused ones, have a genetic component. Furthermore, acceptance of a genome full of antagonistically pleiotropic genetic interactions poses important implications for clinical treatment and disease prevention research, especially genetically based therapies.

Background deleterious effects on their possessors (usually, but not The human genome is the product of an evolutionary always, homozygotes for the mutant allele) relative to history that combines mutational processes and natural those possessing wildtype or normal alleles. Although selection; since this process appears to be a very effec- genetic drift and mutation selection balance can explain tive mechanism for optimizing many traits (i.e., adapta- the presence of segregating allelic variation for deleter- tion) it is reasonable to question why so many genetic ious traits, many genetic disorders are present at fre- disorders (i.e., non-optimal alleles) exist. Genetic disor- quencies higher than we would expect from a historical ders are typically defined as «mutant» alleles that confer process by which mutations arise and are then elimi- nated by selection over time. A potential resolution to the discrepancy between our expectations of very low * Correspondence: [email protected] rates of genetic disorders and our observations of Biology Department, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, CA 90840, USA remarkably common genetic disorders involves

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considering the other effects of these mutant alleles display high penetrance until post-reproduction. A long aside from just the deleterious ones. history of selection for, and fixation of, such antagonisti- In contrast to long-held notions whereby single genes cally pleiotropic alleles is thought to account for bio- encoded single functions (e.g., «the» eye color gene), chemical pathways and physiological processes that most genes are now recognized to have multiple qualita- work well in youth, but less well in older age - i.e., tively distinct functions, a phenomenon termed pleio- senescence [1,3]. tropy [1]. A simple thought experiment where we Previous studies have discussed the influence of senes- consider that the human genome has approximately cence on different human diseases such as atherosclerosis 23,000 genes (with multiple genes involved in the for- [4], sarcopenia [5], osteoporosis [6], immunodeficiency mation of each aspect of our phenotype) encoding a disorders [7] and neurodegenerative disorders such as phenotype with more than 23,000 aspects demonstrates Alzheimer’s([8] (see also a review in [9]). In fact, a num- that many genes must have multiple functions. This ber of alleles associated witharangeofotherdiseases being the case, allelic variants that arise due to mutation may have possessed similar antagonistically pleiotropic maychangetheperformancesofthatgene’sfunctions effects and led to specific aspects of our senescence. For differently such that sometimes these changes result in example, it has been argued that positive selection and opposing effects on fitness - i.e., some mutations can sexual conflict may account for increased carcinoma risk improve the performance of one aspect of a gene’s func- [10]. They reason that higher selective pressures due to tion while simultaneously making another worse. Such a sexual and generational conflict resulted in positive selec- phenomenon is termed antagonistic pleiotropy, the tion, as suggested by relatively high rates of nucleotide pleiotropic functions of the same gene act antagonisti- and amino acid sequence evolution in genes associated cally in terms of the fitness of the possessor. To date, with such conflicts, and that these changes had maladap- consideration of the implications of such antagonistically tive pleiotropic side-effects that increase the rate of carci- pleiotropic effects has largely been the purview of stu- noma [10]. We suggest that in addition to antagonistic dies of senescence. pleiotropy contributing to the fixation of carcinoma caus- Senescence is associated with distinct changes in the ing mutations it may contribute similarly to current poly- physiology of individuals as they age [2]. Natural selec- morphisms of other alleles associated with cancer and tion acts on differences in relative fitness (which is com- other non-cancerous conditions. prised of both mortality and reproduction) in Previous consideration of this topic has tended to individuals within populations; the lack of natural selec- focus on selection that resulted in the fixation of alleles tion to completely eliminate many deleterious senes- that cause their deleterious effects in older age, changing cence-associated traits explains their sustained the default (i.e., wildtype) condition in the population. In prevalence in populations of humans. The most widely actuality, natural selection is often a slow and gradual held evolutionary explanation for the continued pre- process and many alleles that differ in their effect on fit- sence of senescence incorporates the aforementioned ness can be present in populations for prolonged peri- antagonistic pleiotropy; an allele that causes deleterious ods, especially if the overall fitness differences are small. traits can actually be selected to increase in frequency For example, an allele that confers a noticeable disad- over time if the deleterious effects on the fitness of the vantage at one stage of life may also favor the individual possessor are outweighed by other advantageous effects at another stage of life and if the net effect is small then also due to the allele [3]. selection to change the allele’s frequency will be weak Natural selection acts the strongest on traits mani- and the frequency will be changed slowly by selection. fested during an organism’s peak reproductive value, an Natural selection can also act to actively maintain alleles age that corresponds to the beginning of reproduction with individually deleterious effects in populations if in most species (i.e., sexually mature, but young), and they act synergistically to increase fitness when present benefits to an organism during this time increase overall together (e.g., via heterosis or epistatic interactions). fitness more than those manifested at other ages [1]. In A clear example of a medically relevant antagonisti- contrast, natural selection is the weakest on traits mani- cally pleiotropic polymorphic allele was recently fested after the majority of an organism’s reproduction described [11]. They conducted a cohort study of 99 is complete (i.e., middle-aged or elderly), and detriments individuals in Ecuador with Laron Syndrome, a rare to an organism during this time reduce overall fitness form of Dwarfism. Individuals with Laron Syndrome less than those manifested at earlier ages. Pleiotropically may possess one of three different genotypes relating to antagonistic alleles that provide a net increase in fitness their Growth Hormone Receptor gene (GHR); most therefore tend to be those with benefits expressed at a individuals carry an A to G splice site mutation in posi- young age, favoring either fertility or survival, while the tion 180 in exon 6 while some carry a nonsense muta- detrimental phenotypes are not expressed or do not tion designated as R43X and others are heterozygous for Carter and Nguyen BMC Medical Genetics 2011, 12:160 Page 3 of 13 http://www.biomedcentral.com/1471-2350/12/160

these two mutations. That study followed Laron Syn- disease causing alleles. We would also like to stress that, drome patients and their non-dwarf kin from 1988 to due to the lack of attention paid to the potential benefits 1998 and recorded medical information. The study of alleles associated with different pathologies, we found that patients with Laron Dwarfism experienced believe that our collection of examples may only touch virtually no incidences of cancer whereas their non- the surface of the phenomenon and is a conservative Dwarf kin experienced a 17% cancer mortality rate. estimate of its prevalence. After describing a number of Laron dwarfs also seemed to be protected from diabetes; conditions in detail (summarized in Tables 1 and 2) we no incidences were reported among Laron patients discuss possible implications of widespread antagonistic whereas their kin suffered a 5% mortality rate. The pre- pleiotropy for current and proposed medical treatments, sence of the various mutant GHR alleles in this popula- clinical trials and research. tion may be due to drift (the population is quite small and relatively isolated), but may also be due to selection Results acting to maintain this allele in the population due to Huntington’s disease and Increased Fecundity the benefits provided. While the allele described in this Huntington’s disease (HD) is a rare, autosomal domi- case is rare and represents a minor overall effect on nant neurodegenerative disorder in which symptoms health and fitness of the wider population, if this general typically manifest at an age that is post-reproductive, in phenomenon of antagonistic pleiotropy is more com- the range of 30-45 years of age [12]. Affected individuals mon it may account for disorders that have more wide- often experience chorea, uncontrolled and involuntary spread impact. The recognition that alleles identified as movements, in addition to a much shorter life-expec- being deleterious may also provide benefits is one that tancy. Onset of Huntington’s disease is attributed to the we believe has been underappreciated in the medical presence of a high number of CAG trinucleotide repeat literature. lengths within the Huntingtin (HTT) gene [12,13]; in The purpose of this investigation is to determine particular, when the number of CAG repeats exceeds whether evidence of balancing selection due to antago- 37, onset of Huntington’s disease symptoms was much nistic pleiotropy, heterosis or epistatic interactions is more likely to occur [14]. seen in alleles associated with increased risk of certain Huntington’s patients show a notable increase in human diseases. As the phenomena of heterosis and fecundity when compared to the unaffected individuals beneficial epistatic interactions are likely to arise from however, and may also experience lower rates of can- the multi-functional nature of genes, we use the term cer. In a study of the relative fitness of affected and antagonistic pleiotropy as a catch-all term to refer to unaffected individuals in Canada via a comparison of alleles that act in any of these manners. If the net rela- the fitness values of 157 Huntington’s patients with tive fitness conferred by pleiotropically antagonistic 170 related wild-type individuals, the mean number of alleles with mixed effects is higher, or even just compar- offspring for the patients was 39% higher than their able, to wildtype individuals then such alleles would unaffected siblings and 18% higher than a set of unre- likely continue to persist in the population. If mutations lated control individuals matched for age and sex [15]. that create such pleiotropic alleles are not extremely That investigation went on to predict that if such fit- rare, widespread antagonistic pleiotropy may explain the ness values as he observed were genuine and were to maintenance and high prevalence of many disease-asso- persist, the disease-associate polymorphisms may dou- ciated alleles. It becomes of great importance therefore ble in frequency in as short as 150 years (roughly six to determine whether antagonistically pleiotropic alleles generations)[15]. are likely to be rare or may be more common than pre- An additional benefit that may be conferred by these viously realized. antagonistically pleiotropic polymorphisms is a reduced risk of cancer [16]. Sorenson et al [17] reported that a Methods higher number of CAG repeats within the HTT allele is In the body of this manuscript, evidence of antagonistic associated with increased p53 activity. P53 is a tumor pleiotropy in several major diseases is presented on a suppressor protein that has a role in inducing apoptosis disease by disease basis. The pleiotropic alleles, their and although apoptosis of neuronal cells may be the benefits and their pathophysiological costs are all briefly major cause of the neuro-degeneration seen in Hunting- outlined. Our review of the research literature supports ton’s patients, a higher level of p53 may also protect the hypothesis that at least some of the genetic factors from tumor growth and cancer development in general. that increase disease severity or risk may also confer The increased CAG repeat length of HD individuals certain reproductive or survival benefits, and therefore may therefore be an explanation for the reduced inci- provides support for the importance of antagonistic dence of cancer seen in a large registry of Huntington’s pleiotropy as a causal mechanism for the persistence of patients [17]. Carter and Nguyen BMC Medical Genetics 2011, 12:160 Page 4 of 13 http://www.biomedcentral.com/1471-2350/12/160

Table 1 Antagonistically pleiotropic disease alleles Deleterious effect (disease) Gene: variation Benefit Increased risk of cancers: ovarian AR: CAG trinucleotide Females: increased fertility via reduced degree of pre-mature ovarian failure, reduced (females), prostate (males). repeat lengths. risk of breast cancer. Males: increased number of viable sperm, reduced risk of Kennedy’s Disease. Huntington’s disease. HTT: CAG trinucleotide Increased fertility, decreased risk of certain cancers. repeat lengths. Sickle cell disease. Hbb: various point Protection against malaria in the heterozygous state. mutations. Beta-thalassemia. HB: various point Protection against malaria in the heterozygous state. mutations Glucose-6-phosphate G6PD: various Protection against malaria. dehydrogenase (G6PD) deficiency. missense mutations. Cystic fibrosis (CF) CFTR: missense Increased fertility. mutation. Increased risk of osteoporosis in ALOX15: A:G intronic Reduced risk of osetoporosis in youth. old age. polymorphism. These diseases are described in more detail in the text, the presence of an advantage in addition to the deleterious effects of the disease is well-supported by detailed genetic or epidemiological information.

Variation in the CAG repeat length within the HTT defective channel in the body leads to diverse symptoms gene therefore seems to exhibit a form of antagonistic involving many organ systems and tissues, mainly due pleiotropy that perfectly illustrates that proposed by [3]. to the buildup of fluids throughout the body. The Presence of more than 37 CAG trinucleotide repeats in backup of fluid contributes to other serious ailments the HTT gene is associated with an increased risk for including increased susceptibility to infections and var- Huntington’s disease, but is also associated with a sub- ious breathing problems [20]. stantial rise in fertility and perhaps reduced cancer risk. Alleles associated with cystic fibrosis may confer certain benefits via overdominance (i.e., heterozygotes have Cystic fibrosis and fertility higher fitness) or epistatic effects however. Data shows Cystic fibrosis (CF) is an autosomal recessive trait that is that individuals in families with members identified as often lethal, though life expectancy has increased over suffering from CF have more offspring than individuals time from early infancy to late-30s due to better medi- from control families [21]. Even though the offspring cation, therapies and more access to medical care [18]. from CF family members experienced higher mortality The mutation that causes CF is a codon deletion in the rates than the offspring of the non-CF family indivi- CFTR gene that results in the loss of phenylalanine duals, CF family individuals still out-reproduced non-CF from the protein product [19]. Loss of phenylalanine family individuals by roughly 22% (p < 0.001)[21]. results in a non-functional trans-membrane chloride Additionally, using a mouse model, an investigation channel, a channel responsible for the movement of conducted by Gabriel and colleagues [22] showed that bodily fluids throughout many different organs and heterozygotes for non-functioning CFTR alleles were organ systems in the body. The presence of this resistant to cholera toxin which suggests that increased

Table 2 Putative antagonistically pleiotropic disease alleles Disease Gene; Variation Putative Benefit Triosephosphate Isomerase TPI Gene: point mutations. Reduction in oxidative stress. Deficiency Tay Sachs HEXA: missense mutations. Protection against tuberculosis. Hemachromatosis HFE Gene: many point mutation. Better iron absorption, increased resistance to typhoid fever and tuberculosis Phenylketonuria PKU: missense mutation. Lower probability of miscarriage Leukemia PTNP11: various expression level Lower probability of hepatocellular carcinoma mutations. Schizophrenia Chromosomal regions 6p22-p24 and Higher relative fertility (in unaffected relatives). 11q21-22. Cancer TNFRSF11B Gene; Various Polymorphisms Increased Bone Density in Females These diseases have been proposed to have antagonistic pleiotropic effects in their etiology, but the evidence is suggestive and preliminary. Carter and Nguyen BMC Medical Genetics 2011, 12:160 Page 5 of 13 http://www.biomedcentral.com/1471-2350/12/160

resistance to cholera may also explain the persistence of health factor for a large proportion of humanity for over CF. These mouse results were similar to results obtained 500 generations and therefore has had sufficient time to from human tissue cultures [23], however that study did change the frequencies of alleles associated with resis- not explicitly demonstrate a fitness benefit to CF hetero- tance [28]. zygotes in humans. Finally, an advantage arising from TheHb-Salleleisperhapsthebestknownandmost decreased levels of sialic acid on cell membrane surfaces widely used example of antagonistic pleiotropy in dis- in CF heterozygotes was proposed [24]. This decrease cussions of the topic in human disease due to the clear could potentially provide increased resistance to influ- deleterious effect in homozygotes combined with a clear enza and other myxo- and paramyxoviruses, although as advantage in heterozygotes. In contrast to the previous above, cell tissue culture data instead of direct compari- examples the advantages and disadvantages of this allele sons of fitness differences between CF heterozygotes are not experienced by the same individuals, but the and other individuals were used to support this hypoth- overall selective forces acting on the allele remain very esis [24]. similar. Over mankind’s evolutionary history (and in the The allele with the deleted CFTR codon therefore present) selection both favors and acts against the Hb-S seems to exhibit antagonistic pleiotropy. The deletion allele simultaneously, resulting in an equilibrium at a leads to serious health issues in homozygotes, but indivi- frequency much higher than that expected for a purely duals likely to be carrying the allele (I.E., families of CF deleterious allele. sufferers) appear to have more offspring and may experience increased resistance to cholera and other Beta-Thalassemia and malaria resistance infectious diseases. Another example of antagonistic pleiotropy in a hemo- globinopathy is the blood disorder Beta-Thalassemia Sickle Cell Anemia and malaria resistance which is caused by a mutation in the beta chains of Sickle cell disease is an autosomal recessive hemoglobi- hemoglobin [29]. Beta-Thalassemia is an autosomal, nopathy commonly found in individuals endemic to tro- incompletely recessive, mutationinwhichindividuals pical and Mediterranean regions, especially Africa [25]. that are homozygous for the mutation often present Sickle cell disease is attributed to a single missense with full disease symptoms (known as Beta-Thalasse- pointmutationintheHemoglobin(Hb)gene,termed mia Major). Beta-Thalassemia Major symptoms include the Hb-S allele. The mutation leads to the replacement anemia, some heart/vascular problems in adults, and of glutamine by valine at position 6 and to the develop- infants and newborns that are often underweight and ment of the characteristic abnormal sickle shaped ery- smaller in size than average children (experiencing the throcytes that are associated with sickle cell anemia. condition termed «failure to thrive») [29]. Although The sickle shape compromises the elasticity of the red heterozygotes for the mutation often present with mild blood cell and causes these cells to obstruct blood flow symptoms of this disorders, they seem to suffer no dif- through narrow vessels, resulting in ischemia and other ference in life expectancy relative to the general popu- serious problems. Although life-expectancy of indivi- lation [30]. duals with the homozygous Hb-S genotype has A study reported that the possession of the Beta-Tha- increased in recent years in developed nations (median lassemia mutation is associated with increased resistance lifespan of 42-48 in the US) [26], for most of human to malaria and that the basis for malarial protection may history (and even now in undeveloped nations) homozy- be due to early childhood exposure to P. vivax resulting gous individuals had very short expected life expectan- in resistance to Plasmodium falciparum [31]. The pro- cies (I.E., high juvenile mortality). In contrast, tection from malaria increases the relative fitness of individuals who are heterozygous for the Hb-S allele do these individuals greatly and is thought to account for not present with full symptoms and suffer minimal the relatively higher frequency of the Beta-Thalassemia hemoglobinopathies. mutation in geographic regions that experience high The Hb-S allele provides an advantage in individuals rates of malaria. heterozygous for the Hb-S allele via increased resistance The Beta-Thalassemia anemia variant of hemoglobin B to malaria, most notably during the ages of 2 to 16 mirrors the case of sickle cell anemia described above months [27]. The presence of such a benefit early in life due to the clear advantage for heterozygotes and the is thought to account for the relatively higher frequency clear deleterious effects in homozygotes. Interestingly, of the sickle cell allele in geographic regions that experi- while the sickle cell and malaria example is more widely ence high rates of malaria. Malaria is estimated to infect known and taught in biology courses, the argument for approximately 400 million individuals and kill over one the role of malaria in the maintenance of the Beta-Tha- million each year [28]. Although the high rate of malaria lassemia mutation via an overdominance based antago- is a relatively recent phenomenon, it has been a major nistic pleiotropy mechanism predates it [31,32]. Carter and Nguyen BMC Medical Genetics 2011, 12:160 Page 6 of 13 http://www.biomedcentral.com/1471-2350/12/160

Glucose-6-Phosphate Dehydrogenase Deficiency and women (OR = 0.442, p = 0.007) but an increased risk in malaria resistance postmenopausal women (OR = 1.727, p = 0.042) [39]. Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency The guanine allele therefore either reduces or increases is a genetic disorder that often results in hemolytic ane- osteoporosis risk depending on which stage of life one is mia because G6PD is essential for the prevention of considering; if we consider the actions of ALOX15 dur- hemolysis due to reactive oxygen species accumulating ing different life stages to bedifferentfunctionsthen in red blood cells during regular cellular metabolism this allele is a clear case of an antagonistically and from exposure to certain environmental stimuli. A pleiotropy. number of observed mutations to the G6PD gene cause a truncated dehydrogenase enzyme that is severely com- Cancers and reproductive traits promised in function and with an estimated 400 million Cancer is a primary cause of genetically caused mortality affected individuals this disorder is one of the most worldwide, with estimates of up to 569,490 deaths in common among enzyme deficiencies [33]. Homozygous 2010 in the US alone [40]. A number of allelic variants (females) and hemizygous (males) individuals manifest for certain genes have been discovered in which the the effects of the recessive mutation which often results genotypes of individuals seem to correlate with risk of in fetal mortality and, later in life, causes hemolytic ane- developing cancer [41]. One of these genes is the andro- mia if red blood cell production cannot be increased gen receptor (AR) gene. The AR gene contains a enough to offset the rate of destruction from hemolysis. repeated CAG trinucleotide sequence and the CAG Current studies show that the life expectancy of affected repeat length influences gene expression, which deter- individuals can be normal with adequate and frequent mines the density of androgen receptors in human treatment [33,34], although this treatment is not avail- epithelial tissue, mainly of the reproductive system. able in much of the world and was not available In females, a shorter CAG repeat length in the AR throughout most of medical history. gene is associated with ovarian cancer. Shorter repeats As is the case for the hemoglobinopathies described of the CAG segment have been associated with an above, G6PD deficiency is associated with protection increased expression of the AR gene in ovarian tissue from malaria. Various alleles that cause G6PD deficiency [42,43] and another study presented a Western Blot have been associated with improved resistance to analysis of tissue cultures as evidence that increased malaria, especially to the most life-threatening cerebral expression of AR gene is associated with ovarian tumor malaria, in hemizygous males and heterozygous females formation [44]. Consistent with this process, women [35,36]. who inherited a shorter repeat of the CAG segment of Although they cause hemolytic anemia, the resistance the androgen receptor allele presented with an earlier to such a widespread and serious disease conferred by age of incidence of ovarian cancer, roughly about 7.2 G6PD mutations may cause the net fitness effect of years earlier compared to a control group [42]. these alleles to be comparable to wildtype fully func- In males, a shorter CAG repeat length in the AR gene tional G6PD alleles. The high prevalence and serious is associated with prostate cancer. As in ovarian tissue, a health consequences of malaria infection may explain shorter CAG repeat length is associated with increased the extremely high number of cases of G6PD estimated expression of the AR gene in prostate tissue [45]. Higher to exist. density of AR in prostate tissue seems to amplify androgen activity, which is expected to increase cell prolifera- ALOX15 gene and Bone Mineral Density tion and prostate tissue growth rates [46]. Increased cell Osteoporosis is a condition in which the bones weaken, proliferation, via hormonal stimulation for example, is making sufferers more prone to fractures in accidents associated with an increase in frequency and malignancy and falls, and poses a serious global health risk. For of prostate cancer [43,47]. Consistent with this process, example, in the US in 2005 there were an estimated Ingles and colleagues reported that males with a shorter 300,000 hip and 550,000 vertebral fractures [37] and in repeat length experienced a two-fold increase in prostate a Canadian cohort study these fractures were associated cancer risk [46]. Similarly, another investigation reported with 419% and 253% increases in mortality over the an association between shorter CAG repeats and pros- next year respectively [38]. Clinical definition of osteo- tate cancer in 587 prostate cancer patients and 588 con- porosis typically includes low bone mineral density trol individuals and that the association was driven by (BMD) measurements. In a study of SNP frequencies the most severe cases [48]. thepresenceofaguanineinsteadofthemorecommon In contrast, shorter repeat CAG lengths within the AR adenine within intron 12 of the 12/15-lipoxygenase gene may provide benefits to females and males. (ALOX15) gene was associated with a reduced risk of In females, shorter repeats may increase reproductive low BMD at the femoral neck in pre-menopausal ability and reduced breast cancer risk. Shorter CAG Carter and Nguyen BMC Medical Genetics 2011, 12:160 Page 7 of 13 http://www.biomedcentral.com/1471-2350/12/160

repeat lengths within the AR gene are associated with a increased reproductive fitness (manifested mainly in potential increase in reproductive fitness via the link youth) via increased viability of sperm, reduced risk of between AR gene expression and follicular development Kennedy’s disease and perhaps increased reproductive during the uterine cycle. Serum androgen activity is opportunities due to phenotypic traits that are more mediated by the androgen receptor and the effects attractive to females. appear additive, implying that a high density of the receptor (such as that caused by increased expression Other putative antagonistically pleiotropic alleles due to shorter repeat lengths) would amplify androgen The examples of antagonistic pleiotropy cited above activity. Androgen activity is associated with follicular demonstrate the widespread nature of this phenomenon growth and maturation and increases the viability of in terms of physiological systems and tissues as well as oocytes, which increases fertility [49]. Consistent with mechanisms of action. The previous examples are based this process is evidence provided by Chaterjee and col- on specifically characterized genes; other possible occur- leagues in which shorter CAG repeat lengths were asso- rences of antagonistic pleiotropy in humans have been ciated with a reduced degree of pre-mature ovarian suggested, based upon correlations and less comprehen- failure [50]. Shorter repeat lengths may also provide an sive data. Further investigation of the proposed cases of advantage via reduced cancer risk; in a study of women antagonistically pleiotropic alleles below seems war- with the BRCA1 allele, shorter CAG repeat lengths ranted in order to round out our understanding of the within the AR gene were associated with reduced risk of pervasiveness of this phenomenon. breast cancer [51]. Recentevidencesuggeststhattheremaybealink In males, shorter CAG repeat lengths within the AR between Triose Phosphate Isomerase (TPI) deficiency, a gene are associated with a potential increase in repro- disease that causes a number of severe symptoms ductive ability, reduced risk of Kennedy’sdiseaseand (chronic hemolytic anemia, cardiomyopathy, susceptibil- perhaps greater sexual attractiveness. There is evidence ity to infection and severe neurological dysfunction), but of an inverse relation between CAG repeat lengths in may also confer oxidative stress resistance. TPI defi- the AR gene and the level of reproductive fitness; on ciency in yeast confers a protective benefit by reducing average, males with a shorter CAG repeat length pre- oxidative stress during cellular metabolism [56], which sented with fewer non-viable, defective sperm [52]. The suggests that humans who are heterozygous for the levelofseverityandtheassociated amount of defective mutant TPI allele may also experience this benefit. sperm were directly correlated to longer repeat lengths However, more definitive evidence involving vertebrate and individuals with shorter repeats experienced models, if not human models, are necessary to further reduced rates of infertility. There is also evidence that a validate this hypothesis. shorter repeat length is associated with increased counts Tay Sachs disease, a severe neurodegenrative disease of viable sperm in adolescent males [53]. Shorter repeat due to a number of different mutations described in the lengths may provide an advantage via reduced risk of HEXA gene which encodes the alpha-subunit of the Kennedy’s disease, a neuromuscular disease similar to lysosomal enzyme beta-N-acetylhexosaminidase, has ALS that only affects males. In a study of 35 unrelated been proposed to owe its surprisingly high frequency to men with Kennedy’s disease and 75 controls [54] all the the benefit provided by protection from tuberculosis affected males had longer CAG repeat lengths. It during the historical process of urbanization [57,58]. appears that a decreased repeat length is associated with Hemochromatosis, a well-known blood disease, has a higher expressions of a range of male- specific traits very clear genetic basis in its manifestation and progres- that may have had (or indeed, still have) roles in mate sion [59] and is often the result of many different poten- acquisition and attraction. tial mutations to different genes with the most common Variation in the CAG repeat length within the AR mutation being the C282Y mutation of the HFE gene. gene therefore seems to exhibit antagonistic pleiotropy Evidence suggests that individuals heterozygous for the in both females and males. In females, shorter CAG C282Y mutation in the HFE gene may experience bene- repeat lengths correlate with increased risk of ovarian fit due to more efficient dietary iron absorption and via cancer, the second leading cause of gynecological cancer increased resistance to typhoid fever and tuberculosis, deaths [55], yet shorter repeats are also associated with diseases that became more prevalent with the rise of increased reproductive fitness via increased viability of urbanization [59]. oocytes and reduced pre-mature ovarian failure as well Phenylketonuria (PKU) may also display evidence of as reduced breast cancer risk. In males, shorter CAG selection by antagonistic pleiotropy. Woolf [60] pre- repeat length is associated with decreased survivorship sented data which suggests that heterozygote females (mostly in older age) due to higher risk of prostate car- may experience a lower chance of mold infection cinogenesis, but shorter repeats are associated with induced miscarriages; potentially an important Carter and Nguyen BMC Medical Genetics 2011, 12:160 Page 8 of 13 http://www.biomedcentral.com/1471-2350/12/160

advantage prior to modern methods of food sanitation higher than normal level of OPG was associated with and storage. Although the PKU causing allele is likely to stronger bones in both men and women [68]. OPG may be purely deleterious today, its historical heterozygote act as a promoter of osteoblast activity by reducing the advantage may account for its current prevalence. The number of activated osteoclasts in the blood. Human sample of PKU individuals referenced in that study was clinical studies have shown that there is an association small and included only individuals in Ireland and Scot- in increased Bone Mineral Density (BMD) in individuals land where mold problems have have been more severe who have higher circulating levels of OPG in their than elsewhere, alternative explanations may be neces- blood plasma. Such costs and benefits associated with sary to explain the global distribution of PKU. the TNFRSF11B gene would be an example of antago- Another putative example involves hepatocellular car- nistic pleiotropy, but further evidence, including the cinoma (HCC), a well-known cancer, and the PTNP11 identification of the specific genetic polymorphism gene. The wild type allele for PTNP11 is proto-onco- responsible, is needed to support this hypothesis. genic and produces the shp2 protein. In its wild-type For these less characterized cases of antagonistic form, PTNP11 aids in the prevention of HCC but may pleiotropy, and other cases not identified yet, the genetic also contribute to the manifestation of leukemia. Cha- mechanism providing the advantage may be straightfor- peau and colleagues showed that mice and human Leu- ward or more complex. The most straightforward kemia cases are associated with a mutation that leads to mechanism is overdominance (heterozygote advantage), theoverexpressionofthePTNP11gene[61].They but incomplete penetrance of the deleterious aspect of investigated mouse models with the PTNP11 allele the alleles or epistatic interactions that differ in different knocked out to simulate under-expression of the gene genetic backgrounds may also be important for the full and the mice were less prone to develop leukemia, but fitness ramifications of disease alleles. The latter two many developed HCC [61]. This data suggests an antag- mechanisms are more subtle and difficult to recognize onistic relationship between the levels of PTNP11 or demonstrate statistically and this may contribute to expression and the development of leukemia and HCC; the unresolved nature of the examples described in this further studies must be performed to properly ascertain section. this same relationship in humans. Given the number of diseases caused by antagonisti- Even psychological disorders may be subject to selec- cally pleiotropic alleles that have been identified to date tion of antagonistically pleiotropic alleles. Schizophrenia and the potential complexity of the mechanisms provid- is believed to have a heritable component with alleles ing the benefits, many more genetic disorders may exist implicated for genetic susceptibility reported to be that have yet to be identified as antagonistically located in chromosomal regions 6p24-p22 [62] and per- pleiotropic. haps 11q21-22[63]. Other studies have shown that although schizophrenic individuals themselves have Discussion reduced reproductive success, their unaffected relatives We have presented a number of examples where antag- often have higher relative fertility [64], indicating a onistic pleiotropy has been demonstrated (Table 1) or potential advantage conferred by alleles associated with proposed (Table 2) for disease causing alleles. We feel schizophrenia when those are present as single copies or that antagonistic pleiotropy may provide a potential in different genetic backgrounds (i.e., different epistatic explanation for the sustained prevalence of many dis- interactions). ease-associated alleles in the human population and may Osteoprogesterin (OPG) is a cytokine receptor protein be a widespread phenomenon. Because individuals who that has a role in malignant tumor progression and possess these alleles may have an equal or comparable modulation of bone density. OPG is encoded by the relative fitness despite their allele’s deleterious effects tumor necrosis factor receptor superfamily member 11B (or the increased risk of experiencing deleterious effects) (TNFRSF11B) gene [65]. Overexpression of the we predict that pathology-associated alleles will continue TNFRESF11B gene has been associated with several to be present in successive generations despite their epithelial cancers [66] and another study has suggested apparent maladaptive nature. that OPG does, in fact, have a modulatory role in tumor Although some of the studies cited predict an increase angiogenesis [67]. Furthermore, Ito et al [66] have sug- in the frequency of these pleiotropic alleles (e.g.,[15]), gest that overexpression is positively correlated with the whether or not the frequencies of these alleles will severity of the associated epithelial cancer (i.e., malig- increase in successive generations is influenced by chan- nancy increases with the concentration of OPG in the ging environmental, medical and social conditions which blood). A high level of OPG has also been shown to alter the negative and positive aspects of genetic fitness decrease the likelihood of bone disease; Samuelson and differences. For example, chelation treatments for Beta- colleagues presented a human clinical study in which a thalassemia have shown great benefit, changing the life Carter and Nguyen BMC Medical Genetics 2011, 12:160 Page 9 of 13 http://www.biomedcentral.com/1471-2350/12/160

expectancy of homozygotes from a few years after birth contributing factor. Malaria is mainly confined to Medi- to as high as 55 years of age [69]. Another example is terranean and tropical regions of the world and such a seen in CF patients where improved medical care in clear geographical distribution allows for a relatively modern times has increased life expectancy of CF indivi- straightforward comparison of allele frequencies in duals from infancy to mid-reproductive age. Even regions likely and unlikely to benefit from malaria pro- though mortality is still usually experienced before the tection. Malaria is also a very common and serious dis- onset of post-reproductive age, this increase in life- order overall, leading public health agencies to collect expectancy can have a rather large impact on the repro- detailed data and provide funding for research related to ductive fitness of CF individuals. With the deleterious understanding this disease. effects on mortality reduced by modern medicine and Recognition of the advantageous effects of other dis- the benefits intact these disease alleles would now have ease alleles may be harder to demonstrate experimen- a higher net advantage and be expected to rise in fre- tally if the advantage provided by the allele does not quency more than would have been possible prior to have a clear and qualitative effect (e.g., if the advantage modern medicinal techniques. is only a reduced risk of a disorder rather than a com- On the other hand, since many of the benefits we plete elimination of a condition). Such advantageous have discussed involve resistance to diseases that mod- effects may not be recognized except in studies with ern medicine will render rarer or more treatable, the large sample sizes in which many aspects of the health benefits may also be reduced and the net change in the of the subjects are recorded. The recent rise of SNP- fitness provided by these antagonistically pleiotropic based association studies may lead to many more dis- alleles is less clear. In fact, because many of the benefits coveries, but only if researchers are measuring a wide we identified involve resistance to pathogens that are range of fitness components. In the ALOX15/BMD now treated effectively, we may expect these advanta- example described above [39], the pleiotropy of the geous effects to be reduced and natural selection to be identified allele was only recognized because the effects more effective at reducing the frequencies of these both involved the measured trait. The increased repro- alleles in the future. This evolutionary process is typi- duction associated with the Huntington’s disease allele cally very slow however, and even if the alleles become was only demonstrable by measuring the number of off- purely deleterious, the elimination of these alleles would spring born to sufferers [15]. take a long time. For these reasons we recommend that many more One readily noticeable feature of the examples of variables be measured when conducting studies of asso- antagonistic pleiotropy we identified is the overabun- ciations between alleles and medical conditions that are dance of advantageous effects involving malaria; we typically included in published analyses. Life history believe that such bias toward examples involving malaria variables such as total reproduction or age at first repro- is due primarily to two major reasons. First, there is a duction are directly related to fitness and advantageous bias in favor of the search for, and recognition of, genetic effects may be made apparent if these values are malaria resistance benefits due to the high profile of recorded. We also suggest that additional health relevant Beta-Thalassemia and Sickle-Cell disease which are both statistics such as blood pressure, cholesterol or BMI be such well-known examples of over-dominance that they measured and included in studies of associations are often taught in the introductory biology curriculum between alleles and medical conditions as they may that medical researchers experience. Such previous reveal otherwise overlooked advantageous aspects of exposure pre-disposes epidemiological researchers to allelic differences. Measurement of additional variables consider malaria foremost when investigating explana- carriestheriskoffalse-positives, but follow up studies tions for the high frequency of disease alleles. For exam- can be used to minimize that risk whereas failure to ple, a recent epidemiological study was able to generate record data leads to missed effects that would not be a highly detailed map of the distribution of malaria discovered later. cases worldwide [28]. In accordance with expectations, Antagonistic pleiotropy may also have been over- the highest prevalence of malaria incidences occurred in looked because the benefits of these alleles may not be Mediterranean and African regions, displaying a 58% large enough to be readily noticeable when compared to prevalence rate in 2010. This is an increase since 2002 the detrimental effects. A brief mathematical illustration from a prevalence rate of 56.9%. We, therefore, expect using standard techniques from population genetics (i.e., the highest frequencies of malaria resistance mutations Hardy-Weinberg style formulations of equilibrium allele to be found in these regions and studies such as this frequencies) demonstrates that even minor benefits can one indicate the level of attention paid to this disorder. result in moderate equilibrium allele frequencies in the Second, the ease with which heterozygous advantage case of overdominance (heterozygote advantage). First, can be demonstrated with these diseases can also be a define two alleles, «A» and «a», as the wildtype and the Carter and Nguyen BMC Medical Genetics 2011, 12:160 Page 10 of 13 http://www.biomedcentral.com/1471-2350/12/160

mutant allele with relative fitness values for the three 100.0% genotypes of w(AA) = 1, w(Aa) = 1+S ,w(aa)=1-S . Reduction in adv del fitness of For example, a value of Sadv = 0.1 would indicate a 10% homozygotes

f for disease allele, higher fitness for heterozygotes whereas a value of S S del 10.0% del = 0.2 indicates a 20% lower fitness for homozygous for (expressed as %) the «a» allele - a value of S =1wouldbeanallele 100% del that is completely lethal in homozygotes. Using the 50% 1.0% terms above, and assuming that reproductive decisions 25% are not biased by assortative mating or other factors 10% disease genotype, disease genotype, f(aa) aside from selection on the alleles, we can derive the fol- o Frequency Equilibrium 0.1% lowing formulae for the equilibrium frequencies of the 0% 5% 10% 15% 20% 25% «a» allele and «aa» genotype: Increase in fitness in heterozygote, Sadv (expressed as %) 2 2 Figure 2 Expected equilibrium frequency of the disease f (a) =Sadv/(2Sadv +Sdel), f (aa) =Sadv /(2Sadv +Sdel) phenotype caused by an overdominant antagonistically Figures 1 and 2 show predicted equilibrium frequen- pleiotropic allele. Equilibrium frequencies for homozygous cies of the homozygous deleterious genotype for a wide disadvantages, Sdel, of 100% (completely lethal or always causing sterility), 50%, 25% and 10% are shown for a range of heterozygous range of realistic parameters. It may seem unlikely that advantageous effects ranging from 0% (no advantage) to 25% (a situations in which the advantages plotted in Figures 1 moderate, but potentially unrecognized, advantage). Note the Log10 and 2 are not well known, but selective advantages as scale on the Y-axis. high as the 25% shown may be unrecognized - the 39% advantage reported for individuals with the Huntington’s Selective benefit is not required for the observed pre- allele relative to their unaffected siblings [15] was not sence of deleterious alleles in a population; purely dele- obvious prior to study [59]. Even in cases in which the terious alleles that confer no advantages persist in heterozygous advantage is minimal (approximately 1%) populations over time due to recurrent mutations coun- and the allele is lethal in the homozygous state, natural tering the process of elimination by selection. Similar selection is expected to act to maintain the alleles and calculations to those above result in predicted equili- result in long-term frequencies of afflicted individuals brium frequencies much smaller however; ranging from higher than 1 in 10,000 instead of acting to remove genotypic frequencies of approximately 0.0001% for them from the population. This conceptual example lethal alleles up to 0.01% for alleles that incur an essen- illustrates the potential for selection to maintain alleles tially unnoticeable 1% fitness reduction. at medically relevant frequencies even while the benefits These calculations lead us to predict that from the conferred may be subtle enough as to be largely unno- wide array of known genetic disorders, it is the ones ticed in a clinical setting. with the highest frequencies that we predict to be most likely to have antagonistically pleiotropic benefits. The process of antagonistic pleiotropy may therefore be 100.000% Increase in fitness in highly relevant for understanding the genetic disorders heterozygotes, 10.000% most commonly experienced (i.e., those that tend to Sadv (expressed as %) result in the highest burden to society). For more com- 1.000% 25% plex cases of antagonistic pleiotropy which do not fit

10% the overdominant model examined here, we would gen- 0.100% 1% erally expect the beneficial aspects of each allele’s

0.010% 0.1% actions to be comparable to the deleterious effect in order to maintain the allele in the long term - this leads disease genotype, disease genotype, f(aa)

Equilibrium Frequency of Frequency Equilibrium 0.001% us to predict that we would see antagonistically pleiotro- 0% 10% 20% 30% 40% 50% 60% 70% 80% 90%

100% pic effects more often for serious medical conditions in Reduction in fitness of homozygotes for which the deleterious effects are more severe. disease allele, S (expressed as %) del For these reasons, rather than being a process that is Figure 1 Expected equilibrium frequency of the disease involved in the etiology of a random subset of all phenotype caused by an overdominant antagonistically pleiotropic allele. Equilibrium frequencies for heterozygous genetic disorders, we expect to see antagonistic pleio-

advantages, Sadv, of 0.1%, 1%, 5% and 25% are shown for a range of tropy overrepresented in the diseases that are the most homozygous deleterious effects, Sdel, ranging from 0% (no common and have the most severe consequences. deleterious effect) to 100% (allele is lethal or homozygotes are Finally, if antagonistic pleiotropy is indeed as wide- always sterile). Note the Log scale on the Y-axis. 10 spread as our reviewed findings and mathematical Carter and Nguyen BMC Medical Genetics 2011, 12:160 Page 11 of 13 http://www.biomedcentral.com/1471-2350/12/160

calculations suggest, the prevalent nature of this phe- alleles at medically relevant frequencies. Based on the nomenon presents two major implications for the availability of this information despite no concerted research and treatment of human genetic disease. research effort to gather it and the mathematical argu- First, identification of pleiotropic alleles that confer ment we feel that such antagonistic pleiotropy may be a disease resistance or other benefits and elucidation of widespread phenomenon. If such pleiotropy is wide- their mechanisms of action may aid medical and patho- spread this opens avenues for the identification of novel physiological research. For example, in the case of Tay physiological pathways by the study of the pleiotropic Sachs, if the hypothesized anti-tuberculosis benefit pro- effects of disease alleles and suggests that caution be vided by this allele is demonstrated then identification taken when designing genetic therapies to eliminate the of the exact cause of this benefit may be useful for the effects of genes lest their beneficial effects be removed development of novel treatments for tuberculosis - a as well. serious need now that more and more drug resistant strains have evolved. Advances in understanding phy- Authors’ contributions siology may also arise from identification of benefits AC conceived the initial project and provided the population genetic unrelated to disease resistance; a full elucidation of the argument. AN obtained the data used and wrote the first draft of the genetic aspects that contribute to the process by which manuscript. Both authors participated in writing of the final manuscript, addressing all revisions and approved the final manuscript. Huntington’s sufferers produce more offspring may prove useful for infertility treatment. Competing interests Second, if antagonistically pleiotropic effects are per- The authors declare that they have no competing interests. vasive then this suggests that we use caution when we Received: 27 May 2011 Accepted: 12 December 2011 design genetically-based treatments for diseases. One Published: 12 December 2011 currently proposed method for treating or preventing cancer is silencing gene expression via interference RNA References 1. Williams GC: Pleiotropy, Natural Selection, and the Evolution of [70,71]; if such mechanisms completely silence the Senescence. Publication of the American Institute of Biological Sciences 1960, expression of an antagonistically pleiotropic allele this 6:332-337. wouldleadtoareductionnot only in the deleterious 2. Crews D: Human Senescence. 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doi:10.1186/1471-2350-12-160 Cite this article as: Carter and Nguyen: Antagonistic pleiotropy as a widespread mechanism for the maintenance of polymorphic disease alleles. BMC Medical Genetics 2011 12:160.

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