BACKGROUND
genomic structural variants , which are deletions, duplications, inversions and translocations of genomic segments, account for the majority of variable base pairs in primate genomes  <cit> .
because of their sheer size, svs can have strong effects on gene function and regulation if they overlap with protein-coding  or regulatory sequences .
indeed, several studies have revealed important roles that svs play in human evolution  <cit>  and adaptation  <cit> .
disruption of a gene’s function by deletion of its coding sequence likely reduces fitness and predisposes humans to several genetic disorders .
consistent with this notion, deletion variants among humans are distributed significantly away from coding sequences  <cit>  and most exonic deletions are found in very low frequencies in human populations  <cit> .
in a recent study, we searched for unusually old deletion variants that affect coding sequences.
specifically, we identified exonic deletions that evolved before human-neanderthal divergence   <cit> .
we surmised that it is unlikely for a loss-of-function gene deletion to be maintained for this long, especially under negative selection.
thus, we hypothesized that a number of these ancient deletion variants have been evolving under balancing selection.
balancing selection has enjoyed a renewed interest in the evolutionary genomics community.
in its most basic form, balancing selection can be thought of as the combination of adaptive forces that maintain variation longer than expected under neutrality  <cit> .
based on the analyses of recently available human and nonhuman primate genomes, several variants have been shown to be evolving under long-term balancing selection in the human-chimpanzee lineage  <cit> .
in addition, multiple instances of balancing selection within the human gene pool have been reported in the last decade .
in this paper, we investigate the evolution of an ancient gene deletion .
this ~32kb deletion variant overlaps  <dig> genes, lce3b and lce3c, which are both involved in skin tissue repair.
we recently showed that this deletion variant is derived in the homo lineage and that the deletion is present in the denisovan genome, but absent in the neanderthal genome  <cit> .
in the same study, we were able to rule out archaic introgression and concluded that incomplete lineage sorting best explains the observed allele sharing at this locus.
the deletion is very common in humans, reaching up to 70% in some european populations.
moreover, this deletion has been strongly associated with psoriasis susceptibility, with odds ratios ranging from  <dig>   to  <dig>   .
however, the adaptive reasons for why this deletion remains in the population are unknown.
RESULTS
the lce3bc deletion spans slightly more than 32kb in the human reference genome and overlaps with two conserved, protein coding genes, lce3b and lce3c .
we manually confirmed that this deletion has been shared with denisovans, but not with neanderthals .
we then determined single nucleotide variants that are in high linkage disequilibrium  with the lce3bc deletion among  <dig> human genomes independent of population ancestry  <cit>  .
using this dataset, we identified a haplotype block that comprises the lce3bc deletion and its flanking sequences extending to  <dig> kb downstream and  <dig> kb upstream .fig.
<dig> genomic location of the lce3bc deletion.
top: location of the lce3bc deletion on chromosome 1q <dig>  .
below: the zoomed-in look at the region harboring the lce3bc deletion.
the red bar shows where the deletion occurs .
the blue bar represents the  <dig>  kb “target”  sequence that we used to conduct the majority of the population genetics analyses in this study
we constructed a maximum likelihood tree of the variation in the region downstream of the lce3bc deletion  among  <dig> modern human haplotypes, as well as denisovan, altai neanderthal, chimpanzee and rhesus macaque haplotypes .
we found a clear separation of two haplogroups  with perfect segregation of haplotypes carrying the deletion and those that do not.
furthermore, the denisovan haplotype, which carries the deletion, clusters with the deleted human haplotypes, while the neanderthal haplotype, which does not carry the deletion, clusters with the non-deleted haplotypes .
this observation provides further support that the lce3bc deletion evolved before human-denisovan split and remains variable since then.fig.
<dig> global haplotype variation of the lce3bc locus and allele frequency of the lce3bc deletion in modern and ancient populations.
a simplified phylogenetic tree based on  <dig> human haplotypes from the  <dig> genomes project.
we constructed the tree using the single nucleotide variants downstream of the deletion .
haplotypes separated into two distinct haplogroups, which segregate into haplotypes that carry the deletion  and those that do not carry the deletion .
frequency pie charts were superimposed to the tree for an overview of the distribution of the major haplotype groups across continental populations .
b frequency distribution of the lce3bc deletion in ancient human genomes.
the x-axis indicates the date of the samples.
the y-axis indicates the allele frequency of the deletion .
the population acronyms are en: early neolithic european; mn: middle neolithic european; ba: bronze age european.
each blue box indicates the ancient population and the length of the box spans the estimated age of the samples.
the sample size of each population is indicated by the red number on the top or bottom of each box.
c the geographic distribution of the lce3bc deletion allele frequency.
the red color indicates the frequency of the haplotypes in each population carrying the deletion, whereas white are haplotypes without the deletion
we then traced back the allele frequency of rs <dig>  which tags the lce3bc deletion in all modern humans , in recently available ancient genomes .
we found that the genome of the  <dig>  year old individual from central asia   <cit>  is heterozygous for the deletion allele .
in fact, we were able to directly confirm the loss of read-depth due to heterozygous deletion in this sample .
we also found that the deletion haplotype has always been found in high frequency in ancient european populations .
the lce3bc deletion is very common in extant human populations as well, reaching major allele status in all non-african populations .
to explain the evolutionary forces that shape this ancient  and very common allele , we consider three scenarios.
first, it is plausible that the lce3bc deletion evolved under neutrality .
indeed, in a recent study, we provided evidence that a neighboring gene, flg, harbors neutrally evolving common loss-of-function variants  <cit> .
in this scenario, we would expect the haplotype block carrying the deletion variant to show no significant deviation in tests of neutrality, when compared to neutral regions of the genome.
second, we considered positive selection, which would increase the frequency of this variant in human populations.
such cases were reported for other loss of function variants  <cit> .
in this scenario, we expect increased population divergence   <cit>  and deviation from expected homozygosity )  <cit> , but reduced nucleotide diversity .
third, we considered balancing selection, where we expect high π and a deviation from the expected frequency site spectrum .
to test the above hypotheses, we compared different population genetics statistics for the lce3bc haplotype block to those calculated for neutrally evolving regions as defined by arbiza et al.
<cit>  .
we found no significant increase of ∆ihh or fst in the lce3bc haplotype block as compared to neutral regions .
in contrast, we observed that nucleotide diversity  in the lce3bc haplotype block is at least  <dig> fold higher than neutral regions   similarly, tajima’s d statistics in the lce3bc haplotype block was significantly higher than neutral regions in all populations tested  .
tajima’s d compares the pairwise differences between haplotypes and the number of segregating sites  <cit> .
in practice, positive values of tajima’s d indicate an excess of intermediate frequency variants, which is a hallmark of balancing selection.
last but not least, we found that the region 10kb downstream of the lce3bc deletion is within the 10th percentile of genome-wide distribution of pairwise time to most recent common ancestor as measured by tsel method  <cit> .table  <dig> summarized mean and standard deviation values for tajima’s d and pi for different population and for the target and neutral regions, as well as regions adjacent to comparable nonexonic ancient deletions
fig.
<dig> population genetics summary statistics  y-axis of the boxplot shows the nucleotide diversity  and  tajima’s d in three different populations .
the red boxes represent all regions of the genome that are predicted to be evolving under neutrality, whereas the blue boxes represent the data points from the lce3bc haplotype block.
the green boxes represent π and tajima’s d scores calculated for the size-matched downstream regions of non-exonic ancient deletions.
these deletions are comparable to the lce3bc deletion in the sense that they were also found to have evolved before human neanderthal/denisovan divergence and that they are similarly high in allele frequency  <cit> .
p-values  are shown on top of the comparisons
these standard tests of neutrality can be affected by the mutation rate, as well demographic history  <cit> .
one specific worry was whether preselecting a known “ancient” deletion that is shared with an archaic hominin species may bias our results.
for example, it is possible that increased π may be due to the fact that the variation in this locus is older than many other parts of the genome.
to address this, we compared tajima’s d and π observed for the lce3bc haplotype block with those calculated for size matched regions downstream of  <dig> other “ancient”, non-exonic deletions.
these deletions were previously found to be shared with neanderthals and/or denisovans due to incomplete lineage sorting and also present in high allele frequency in contemporary human populations.
as such, they are comparable to the lce3bc deletion both for their age and allele frequency.
this analysis confirmed our previous observations that π  and tajima’s d  calculated for the lce3bc haplotype block is significantly higher than those calculated for other ancient deletion haplotype blocks .
we also considered the potential impact of repetitive sequences to variant calling quality.
as such, we confirmed all our main results, omitting variants coinciding with repetitive sequences .
discussion
in this study, we analyzed the haplotype block specifically because it harbors an ancient, exonic and disease-associated deletion variant.
our analyses of the haplotypic variation harboring this deletion best fits a model where the lce3bc deletion has been maintained under balancing selection in human populations.
what remains an open question is the adaptive pressure on the lce3b and lce3c gene functions at the organismal and ecological levels.
the phenotypic impact of the lce3bc deletion has been discussed extensively, especially within the context of psoriasis biology .
briefly, lce3b and lce3c coding sequences, which remain highly conserved across mammals , are both active in skin barrier repair.
as such, their expression is mostly confined to injured skin  <cit>  and deletion of these genes likely leads to inefficient skin barrier repair.
in addition, a likely regulatory region is also eliminated by the deletion  <cit>  .
it also appears that the deletion haplotype leads to a significant increase in lce3a expression in sun-exposed skin , which may be a partial compensatory response to loss of lce3b and lce3c activity.
it is unknown how the lack of lce3b and lce3c activity due to their deletion leads to psoriasis susceptibility.
variants that are related to skin structure  and variants related to immune function  are independently associated with psoriasis.
moreover, epistatic interactions between hla-c* <dig> and lce3bc loci were reported within the context of psoriasis  <cit> .
briefly, it appears that the lce3bc deletion leads to slower repair of the epidermal barrier, which in turn leads to increased exposure to environmental antigens and pathogens.
the higher level of exposure consequently leads to higher activity of immune elements, and occasionally pathological autoimmune response.
based on the above-described mechanism, bergboer et al.
<cit>  hypothesized that the lce3bc deletion would be favored to increase the effectiveness of the acquired immunity system , with the drawback of increasing the susceptibility to autoimmune disorders.
in fact, it is plausible that the effect of the lce3bc deletion on the immune system may be more than skin-deep, as the deletion was also associated with more systemic autoimmune disorders, such as psoriatic arthritis  <cit>  and lupus  <cit> .
our findings presented here are concordant with this hypothesis.
CONCLUSIONS
the recent availability of high quality whole genome data at the population level provides novel opportunities to investigate complex evolutionary forces that shape disease susceptibility loci without ascertainment bias.
using such an approach, we provide multiple lines of evidence that a common, 32kb deletion strongly associated with psoriasis has evolved under balancing selection in the human lineage  <cit> .
our study presents empirical evidence that balancing selection on the lce3bc deletion contributes to this very interesting dynamic.
our results will also contribute to the renewed discussion in the community on balancing selection maintaining advantageous diversity in human populations  <cit> .

 genomic structural variants, which are deletions, duplications, inversions and translocations of genomic segments, account for the majority of variable base pairs in primate genomes. because of their sheer size, svs can have strong effects on gene function and regulation if they overlap with protein-coding or regulatory sequences. indeed, several studies have revealed important roles that svs play in human evolution and adaptation. disruption of a gene’s function by deletion of its coding sequence likely reduces fitness and predisposes humans to several genetic disorders. consistent with this notion, deletion variants among humans are distributed significantly away from coding sequences and most exonic deletions are found in very low frequencies in human populations. in a recent study, we searched for unusually old deletion variants that affect coding sequences. specifically, we identified exonic deletions that evolved before human-neanderthal divergence. we surmised that it is unlikely for a loss-of-function gene deletion to be maintained for this long, especially under negative selection. thus, we hypothesized that a number of these ancient deletion variants have been evolving under balancing selection. balancing selection has enjoyed a renewed interest in the evolutionary genomics community. in its most basic form, balancing selection can be thought of as the combination of adaptive forces that maintain variation longer than expected under neutrality. based on the analyses of recently available human and nonhuman primate genomes, several variants have been shown to be evolving under long-term balancing selection in the human-chimpanzee lineage. in addition, multiple instances of balancing selection within the human gene pool have been reported in the last decade. in this paper, we investigate the evolution of an ancient gene deletion. this ~32kb deletion variant overlaps genes, lce3b and lce3c, which are both involved in skin tissue repair. we recently showed that this deletion variant is derived in the homo lineage and that the deletion is present in the denisovan genome, but absent in the neanderthal genome. in the same study, we were able to rule out archaic introgression and concluded that incomplete lineage sorting best explains the observed allele sharing at this locus. the deletion is very common in humans, reaching up to 70% in some european populations. moreover, this deletion has been strongly associated with psoriasis susceptibility, with odds ratios ranging from to. however, the adaptive reasons for why this deletion remains in the population are unknown.  the lce3bc deletion spans slightly more than 32kb in the human reference genome and overlaps with two conserved, protein coding genes, lce3b and lce3c. we manually confirmed that this deletion has been shared with denisovans, but not with neanderthals. we then determined single nucleotide variants that are in high linkage disequilibrium with the lce3bc deletion among human genomes independent of population ancestry. using this dataset, we identified a haplotype block that comprises the lce3bc deletion and its flanking sequences extending to kb downstream and kb upstream.fig. genomic location of the lce3bc deletion. top: location of the lce3bc deletion on chromosome 1q. below: the zoomed-in look at the region harboring the lce3bc deletion. the red bar shows where the deletion occurs. the blue bar represents the kb “target” sequence that we used to conduct the majority of the population genetics analyses in this study we constructed a maximum likelihood tree of the variation in the region downstream of the lce3bc deletion among modern human haplotypes, as well as denisovan, altai neanderthal, chimpanzee and rhesus macaque haplotypes. we found a clear separation of two haplogroups with perfect segregation of haplotypes carrying the deletion and those that do not. furthermore, the denisovan haplotype, which carries the deletion, clusters with the deleted human haplotypes, while the neanderthal haplotype, which does not carry the deletion, clusters with the non-deleted haplotypes. this observation provides further support that the lce3bc deletion evolved before human-denisovan split and remains variable since then.fig. global haplotype variation of the lce3bc locus and allele frequency of the lce3bc deletion in modern and ancient populations. a simplified phylogenetic tree based on human haplotypes from the genomes project. we constructed the tree using the single nucleotide variants downstream of the deletion. haplotypes separated into two distinct haplogroups, which segregate into haplotypes that carry the deletion and those that do not carry the deletion. frequency pie charts were superimposed to the tree for an overview of the distribution of the major haplotype groups across continental populations. b frequency distribution of the lce3bc deletion in ancient human genomes. the x-axis indicates the date of the samples. the y-axis indicates the allele frequency of the deletion. the population acronyms are en: early neolithic european; mn: middle neolithic european; ba: bronze age european. each blue box indicates the ancient population and the length of the box spans the estimated age of the samples. the sample size of each population is indicated by the red number on the top or bottom of each box. c the geographic distribution of the lce3bc deletion allele frequency. the red color indicates the frequency of the haplotypes in each population carrying the deletion, whereas white are haplotypes without the deletion we then traced back the allele frequency of rs which tags the lce3bc deletion in all modern humans, in recently available ancient genomes. we found that the genome of the year old individual from central asia is heterozygous for the deletion allele. in fact, we were able to directly confirm the loss of read-depth due to heterozygous deletion in this sample. we also found that the deletion haplotype has always been found in high frequency in ancient european populations. the lce3bc deletion is very common in extant human populations as well, reaching major allele status in all non-african populations. to explain the evolutionary forces that shape this ancient and very common allele, we consider three scenarios. first, it is plausible that the lce3bc deletion evolved under neutrality. indeed, in a recent study, we provided evidence that a neighboring gene, flg, harbors neutrally evolving common loss-of-function variants. in this scenario, we would expect the haplotype block carrying the deletion variant to show no significant deviation in tests of neutrality, when compared to neutral regions of the genome. second, we considered positive selection, which would increase the frequency of this variant in human populations. such cases were reported for other loss of function variants. in this scenario, we expect increased population divergence and deviation from expected homozygosity ), but reduced nucleotide diversity. third, we considered balancing selection, where we expect high π and a deviation from the expected frequency site spectrum. to test the above hypotheses, we compared different population genetics statistics for the lce3bc haplotype block to those calculated for neutrally evolving regions as defined by arbiza et al.. we found no significant increase of ∆ihh or fst in the lce3bc haplotype block as compared to neutral regions. in contrast, we observed that nucleotide diversity in the lce3bc haplotype block is at least fold higher than neutral regions similarly, tajima’s d statistics in the lce3bc haplotype block was significantly higher than neutral regions in all populations tested. tajima’s d compares the pairwise differences between haplotypes and the number of segregating sites. in practice, positive values of tajima’s d indicate an excess of intermediate frequency variants, which is a hallmark of balancing selection. last but not least, we found that the region 10kb downstream of the lce3bc deletion is within the 10th percentile of genome-wide distribution of pairwise time to most recent common ancestor as measured by tsel method.table summarized mean and standard deviation values for tajima’s d and pi for different population and for the target and neutral regions, as well as regions adjacent to comparable nonexonic ancient deletions fig. population genetics summary statistics y-axis of the boxplot shows the nucleotide diversity and tajima’s d in three different populations. the red boxes represent all regions of the genome that are predicted to be evolving under neutrality, whereas the blue boxes represent the data points from the lce3bc haplotype block. the green boxes represent π and tajima’s d scores calculated for the size-matched downstream regions of non-exonic ancient deletions. these deletions are comparable to the lce3bc deletion in the sense that they were also found to have evolved before human neanderthal/denisovan divergence and that they are similarly high in allele frequency. p-values are shown on top of the comparisons these standard tests of neutrality can be affected by the mutation rate, as well demographic history. one specific worry was whether preselecting a known “ancient” deletion that is shared with an archaic hominin species may bias our results. for example, it is possible that increased π may be due to the fact that the variation in this locus is older than many other parts of the genome. to address this, we compared tajima’s d and π observed for the lce3bc haplotype block with those calculated for size matched regions downstream of other “ancient”, non-exonic deletions. these deletions were previously found to be shared with neanderthals and/or denisovans due to incomplete lineage sorting and also present in high allele frequency in contemporary human populations. as such, they are comparable to the lce3bc deletion both for their age and allele frequency. this analysis confirmed our previous observations that π and tajima’s d calculated for the lce3bc haplotype block is significantly higher than those calculated for other ancient deletion haplotype blocks. we also considered the potential impact of repetitive sequences to variant calling quality. as such, we confirmed all our main results, omitting variants coinciding with repetitive sequences. discussion in this study, we analyzed the haplotype block specifically because it harbors an ancient, exonic and disease-associated deletion variant. our analyses of the haplotypic variation harboring this deletion best fits a model where the lce3bc deletion has been maintained under balancing selection in human populations. what remains an open question is the adaptive pressure on the lce3b and lce3c gene functions at the organismal and ecological levels. the phenotypic impact of the lce3bc deletion has been discussed extensively, especially within the context of psoriasis biology. briefly, lce3b and lce3c coding sequences, which remain highly conserved across mammals, are both active in skin barrier repair. as such, their expression is mostly confined to injured skin and deletion of these genes likely leads to inefficient skin barrier repair. in addition, a likely regulatory region is also eliminated by the deletion. it also appears that the deletion haplotype leads to a significant increase in lce3a expression in sun-exposed skin, which may be a partial compensatory response to loss of lce3b and lce3c activity. it is unknown how the lack of lce3b and lce3c activity due to their deletion leads to psoriasis susceptibility. variants that are related to skin structure and variants related to immune function are independently associated with psoriasis. moreover, epistatic interactions between hla-c* and lce3bc loci were reported within the context of psoriasis. briefly, it appears that the lce3bc deletion leads to slower repair of the epidermal barrier, which in turn leads to increased exposure to environmental antigens and pathogens. the higher level of exposure consequently leads to higher activity of immune elements, and occasionally pathological autoimmune response. based on the above-described mechanism, bergboer et al. hypothesized that the lce3bc deletion would be favored to increase the effectiveness of the acquired immunity system, with the drawback of increasing the susceptibility to autoimmune disorders. in fact, it is plausible that the effect of the lce3bc deletion on the immune system may be more than skin-deep, as the deletion was also associated with more systemic autoimmune disorders, such as psoriatic arthritis and lupus. our findings presented here are concordant with this hypothesis.  the recent availability of high quality whole genome data at the population level provides novel opportunities to investigate complex evolutionary forces that shape disease susceptibility loci without ascertainment bias. using such an approach, we provide multiple lines of evidence that a common, 32kb deletion strongly associated with psoriasis has evolved under balancing selection in the human lineage. our study presents empirical evidence that balancing selection on the lce3bc deletion contributes to this very interesting dynamic. our results will also contribute to the renewed discussion in the community on balancing selection maintaining advantageous diversity in human populations. 
our results are most parsimonious with the scenario where the lce3bc deletion has evolved under balancing selection in humans. here, we show that the haplotype block that harbors the deletion retains high allele frequency among extant and ancient human populations; harbors unusually high nucleotide variation; contains an excess of intermediate frequency variants; and has an unusually long time to coalescence to the most recent common ancestor. a common, 32kb deletion of lce3b and lce3c genes is strongly associated with psoriasis. however, it was not clear why negative selection has not removed this deletion from the population.
 a common, 32kb deletion of lce3b and lce3c genes is strongly associated with psoriasis. we recently found that this deletion is ancient, predating human-denisovan divergence. however, it was not clear why negative selection has not removed this deletion from the population.  here, we show that the haplotype block that harbors the deletion retains high allele frequency among extant and ancient human populations; harbors unusually high nucleotide variation; contains an excess of intermediate frequency variants; and has an unusually long time to coalescence to the most recent common ancestor.  our results are most parsimonious with the scenario where the lce3bc deletion has evolved under balancing selection in humans. more broadly, this is consistent with the hypothesis that a balance between autoimmunity and natural vaccination through increased exposure to pathogens maintains this deletion in humans. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords copy number variationgenomic structural variantsatopic dermatitishladefensinsneanderthallce3ahuman evolutionhttp://dx.doi.org/ /100008209university at buffalostart-up fundsgokcumen omer issue-copyright-statement© the author 2016 