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Post by Admin on Oct 29, 2019 18:18:23 GMT
Fat, Vitamin D, and the Mammary Gland Given that vitamin D is intricately intertwined with adipose physiology, it is not surprising that vitamin D plays a role in virtually all aspects of mammary gland function, as evinced by the presence and activation of the Vdr in mesenchymal and epithelial breast tissue (98, 99). Vitamin D also appears to influence ductal branching and, with it, milk content. There are three distinct phases of breast development (Fig. 2B): (i) hormone-independent embryonic development during which buds form, sprout, and start to branch into the precursor of the fatty stroma to form a ductal tree; (ii) the onset of pubertal hormonal signaling when terminal end buds form at the tips of the mammary ducts and start to invade the fat pad from which they will extract milk fats, vitamins, and other nutrients (100⇓–102); and (iii) the repeatable hormone-induced cycles of pregnancy, lactation, and weaning (101, 102). Vitamin D-deficient adipose tissue has been modeled by an adipose-specific Vdr knockout (Vdr KO) mouse, revealing significant and, from an evolutionary perspective, fascinating sex-by-diet effects (99). Female mice that are unable to bioactivate vitamin D in adipocytes exhibit increased visceral adipose tissue overall, but they have a normal mammary fat pad mass. Although the mass of the mammary fat pad is unaffected by the loss of Vdr, there is an increase in the ductal branching density within it (99). Surprisingly, however, this only occurred when the mice are fed a high-fat diet (reminiscent of traditional human diets in Artic cultures) (99). These results suggest that ductal branching is stimulated by adjacent vitamin D-deficient adipose tissue in response to high-fat diets during the hormonally induced stages of mammary development (98, 99, 103). In sharp contrast to the results for female mice, there are no effects in males with the adipose-specific Vdr deletion in either the normal or high-fat diet (99). The results of the Vdr KO study, bolstered by additional research (reviewed in ref. 73), reveal a sex-specific relationship between vitamin D deficiency and adipose tissue that includes an increase in mammary ductal branching during the hormone-induced stages of breast development (Fig. 2B). This physiological increase in ductal branching in response to very low levels of vitamin D indicates that other genetic mechanisms that increase ductal branching may be positively selected for in populations experiencing chronic vitamin D deficiency, such as in UV-poor environments at high latitudes. A completely independent line of evidence suggests that variation in ductal branching influences milk content (Fig. 2A). Variation in milk vitamin D levels is heritable in cows and shows evidence of positive selection over the short time frames associated with animal domestication, demonstrating that the underlying genetic mechanisms are highly responsive to selective pressure (104, 105). Milk vitamin D content varies by cattle breed, with some producing notably higher concentrations irrespective of UV exposure (reviewed in ref. 106). Functional associations of 31 genes expressed in cows producing different milk protein and fat concentrations include transcriptomes of genes involved in mammary gland bud elongation (107), suggesting that mammary duct branching contributes to the genetic underpinnings of variation in milk composition. EDAR V370A’s Influence on Ductal Branching The relationship between vitamin D and ductal branching calls for a renewed consideration of EDAR V370A’s influence on mammary duct branching density as the phenotypic target of selection. As with other ectodermally derived structures (e.g., teeth), the earliest phase of mammary development results from epithelial–mesenchymal tissue interactions at embryonic day 11 in the mouse (100⇓–102). Ductal branching is established via NF-ĸB signaling, a downstream target of the ectodysplasin pathway (108). The increase in branching observed in mouse models of EDAR V370A (30, 31, 44) is likely the result of the EDA/EDAR/NF-ĸB pathway’s influence on the proteins that modulate the strength of the junctions that adhere the myoepithelial cells of the mammary ducts (109⇓–111) (Fig. 2 C–E), facilitating or hindering branching during morphogenesis (112). The more elaborate ductal branching induced by EDAR V370A during the embryonic stage of mammary development likely enhances the physiological effect of the increased branching induced by vitamin D deficiency in the later stages of breast development (during puberty and gestation/lactation). Conclusions The Arctic is an extreme environment for humans because of the almost complete lack of UV-B exposure that is required for the biosynthesis of vitamin D. This lack of vitamin D would have reduced immunological function and bone development and hindered the healthy function of adipose tissue. Here, we have presented a diverse array of evidence supporting the hypothesis that a genetically isolated population living in this environment during the LGM ∼20,000 y ago experienced selection for polymorphisms in the FADS gene cluster and for EDAR V370A because of the advantage these genetic variants likely confer in transmitting nutrients from mother to infant through breast milk under conditions of extremely low UV. PNAS May 8, 2018 115 (19) E4426-E4432; first published April 23, 2018
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Post by Admin on Nov 24, 2019 6:13:59 GMT
Humans expanded within and out of Africa between 50,000 and 100,000 years ago and now inhabit radically different physical and cultural environments around the globe. A long-standing question in anthropology has been: How have humans genetically adapted to these environments? Numerous recent studies have attempted to address this issue for a review, see [1]. Most of these studies aim to identify genomic regions that have experienced local positive selection (i.e. geographically-restricted positive selection) from genome-wide polymorphism data e.g. [2], [3], [4]. A small number of these genomic regions are associated with phenotypes that have long been candidates for local positive selection, such as lactose tolerance [5], [6] and skin pigmentation [7]–[12]. For the large majority of these regions, however, no association to a locally adaptive phenotype has been established and the precise location of functional adaptive variants remains elusive. Moreover, sexual selection may be a plausible explanation for at least some of these candidate regions. Further investigation of candidate regions, including the possible functional consequences of the genetic variation observed in such regions, is required to fully understand the impact of local positive selection on our species. Here, we investigate one candidate gene, EDAR, in detail. A strong signature of positive selection in East Asians has been found in the genomic region containing the EDAR gene (HGNC∶2895) according to tests based on the allele frequency spectrum [4], [13], [14], haplotype structure [2], [3] and population differentiation [15]–[19]. EDAR is known to be involved in the development of hair follicles, teeth and sweat glands [20] and harbors a nonsynonymous single nucleotide polymorphism (SNP) (rs3827760) that results in a valine to alanine substitution at position 370 of the amino acid sequence (V370A). Here we test the function of V370A in vitro and provide several lines of evidence suggesting that there was positive selection on the derived 370A allele in East Asians prior to 10,000 years ago. Figure 1. Worldwide allele frequencies and population differentiation for V370A. The vertical bar chart displays the frequency of the 370A allele in each of the populations represented in the CEPH-HGDP panel with sample sizes (number of individuals) on the left. The shaded boxes in the 53×53 and 7×7 matrices show which pairwise Fst values are significant compared to the empirical distribution at three P value thresholds (see the boxed-in P value legend). Results Fst analysis We assessed worldwide population differentiation for V370A by use of the Fst statistic [21]. Alleles that have been targets of local positive selection tend to have unusually high Fst values [22]–[24]. We genotyped the V370A polymorphism in the 53 worldwide populations of the CEPH Human Genome Diversity Panel CEPH-HGDP; [25] and compared Fst values for V370A to an empirical Fst distribution derived from 2750 autosomal markers (2540 SNPs [26] and 210 indels [27]) previously typed in the same set of samples. Global Fst, the degree of differentiation among all 53 populations, for V370A is 0.760. This value is higher than all Fst values from the empirical global Fst distribution. To examine the patterns of population differentiation at a more refined geographical scale, we calculated Fst for every pairwise comparison among the 53 populations and 7 geographic regions to produce 53×53 and 7×7 Fst matrices, respectively. Each pairwise Fst value for V370A was then compared to the corresponding empirical distribution of pairwise Fst values to generate a P value. Worldwide allele frequencies and the P value matrices from the Fst analysis are depicted in Figure 1. Figure 2. Multiple species alignment of the death domain of EDAR. The derived 370A allele is shaded in dark grey. Variable positions in the alignment are shaded in light grey. Sites at which nonsynonymous substitutions are known to result in hypohidrotic ectodermal dysplasia are boxed in. An asterisk indicates the site at which an Arg-His substitution causes decreased activation of NF-κB [28]. Functional assay EDAR (Swiss-Prot∶Q9UNE0) is a cell-surface receptor that, upon binding to its ligand, induces an intracellular cascade leading to the activation of NF-κB, a transcription factor [20]. To investigate the functional consequences of the V370A polymorphism, we measured NF-κB activation in vitro using a luciferase reporter assay, from a HEK293 cell line heterologously expressing the 370V and 370A variants of EDAR cDNA. As a positive control, we also performed the same transfections with 370V and 370A EDAR cDNAs that harbored an additional disease mutation (375H; Figure 2) that had previously been shown to severely reduce NF-κB activation in vitro [28]. In agreement with previous work, the cDNAs carrying 375H showed a ∼6 fold reduction in activation of NF-κB compared to cDNAs without 375H (Figure 3). Moreover, the derived 370A allele results in increased activation of NF-κB compared to the ancestral 370V allele on both the normal (p = 0.018, univariate ANOVA with experiment as a random factor and clone as a fixed factor) and the disease background (p<0.004, two-tailed t-test assuming equal variances; Figure 3).
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Post by Admin on Nov 24, 2019 21:39:17 GMT
Figure 3. The derived 370A allele of EDAR results in enhanced activation of NF-κB in vitro. Transfection of a HEK293 cell line with ancestral (370V) and derived (370A) versions of EDAR activate NF-κB to a different extent on both a normal genetic background (comparison on the left) and on a background containing a known disease mutation (375H; comparison on the right). Luciferase activity is driven by EDAR-activated NF-κB. Data was averaged from two independent experiments with reads from at least 9 wells total for each clone. Error bars are standard error of the mean (* p<0.05, ** p<0.01). Estimation of time since fixation Finally, we assessed the timing of the selection event on 370A from ∼22 kb of EDAR sequence from 23 individuals of Chinese ancestry from the Seattle SNPs data set (http://pga.gs.washington.edu/). We employed a method that estimates the time since fixation of a beneficial allele [29]. Only one chromosome from the data set carried the ancestral 370V allele and we removed it from the analysis and assumed that the 370A allele had reached fixation. The resulting estimate for the time since fixation is 10,740 years (95% CI = 1133–73996; Figure 4). Figure 4. Density plot of the posterior distribution of estimates of the time since fixation of the 370A allele. The estimated time since fixation of the 370A allele is the mode of the distribution (10740 years before present). The 2.5% and 97.5% confidence intervals are boxed in and are indicated by the hashed vertical lines.
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Post by Admin on Nov 25, 2019 20:36:08 GMT
Discussion Recent scans of genome-wide polymorphism data have generated long lists of genomic regions that are believed to have been targeted by local positive selection, but few of these regions have been shown to harbor functional variants or to be linked to a putatively adaptive phenotype. Detailed investigation of these candidate genomic regions is required for a comprehensive picture of local human adaptation. The present study provides an analysis of genetic variation and function at the EDAR gene, a candidate for positive selection in East Asians identified from recent genome scans [2]–[4], [13]–[16], [19].
We provide two lines of evidence supporting the hypothesis that the derived 370A allele in EDAR is functional and experienced positive selection in East Asians. First, worldwide population differentiation for 370A as measured by Fst is highly unusual. Figure 1 demonstrates that 370A has a highly unusual worldwide frequency distribution, supporting a scenario in which the 370A allele was driven to high frequency in East Asians and Native Americans by positive selection (Figure 1). Two Central/South Asia populations, the Uygur and the Hazara, have intermediate frequencies of 370A (0.44 and 0.5, respectively), in agreement with their close genetic relationship to East Asians [30], [31]. The 370A allele is also found at low frequency in Melanesia (0.12), and was likely introduced there via the recent Austronesian expansion [32]. Otherwise 370A is absent in Africans and Papuans and is observed at very low frequency in most of Central/South Asia, Europe and the Middle East (Figure 1).
The second line of evidence suggesting an adaptive functional role for 370A stems from its location in the amino acid sequence of EDAR. V370A is located in the death domain, a protein interaction module, of EDAR. The death domain of EDAR is highly conserved (Figure 2) and interacts with the death domain of EDARADD, an intracellular ligand to EDAR [33], [34]. This interaction initiates an intracellular signalling cascade that results in the activation of the transcription factor NF-κB [35]. Therefore, V370A may alter binding affinity with the death domain of EDARADD and thereby influence the activation of NF-κB. Moreover, seven nonsynonymous substitutions in the death domain of EDAR cause hypohidrotic ectodermal dysplasia (HED) in humans [36], a disease characterized by sparse and thin hair, missing teeth and the absence of sweat glands (OMIM∶604095; see Figure 2). In particular, the R375H substitution, only 5 amino acids upstream from V370A, results in a loss of affinity for EDARADD and reduced NF-κB activation [28]. We therefore tested the function of V370A in vitro and found that the 370A allele does differ from the ancestral 370V allele in that it results in enhanced NF-κB activation (Figure 3).
Our results contradict a recent report from Fujimoto et al. [37] in which the 370A allele was shown to reduce NF-κB activation in vitro [37]. We are confident that our results are correct for two reasons. First, in addition to observing enhanced NF-κB activation for the derived 370A allele on a normal genetic background, we measured NF-κB activation of the 370V and 370A alleles on the background of a disease mutation (375H) that was previously demonstrated to result in significantly reduced NF-κB activation [28]. In agreement with [28], we observed significantly reduced NF-κB activation in both clones carrying the 375H disease mutation. Moreover, NF-κB activation was significantly higher in the derived 370A+375H construct than in the ancestral 370V+375H construct (Figure 3). Thus, we observed enhanced NF-κB activation for the derived 370A allele in two independent constructs. Second, East Asians have thicker hair than Europeans and Africans [38] and an increase in NF-κB activation is arguably more likely to lead to the thicker East Asian hair phenotype. This is because a decrease in NF-κB activation, as observed for carriers of the 375H allele that causes hypohidrotic ectodermal dysplasia, is associated with thin hair. We suspect that the large doses of plasmid DNA (300 ng) and long post-transfection incubation period (48 h) could have induced cell death in the experiments of Fujimoto et al. [37]. Although EDAR-induced cell death is a matter of controversy [39], [40], several features of cell death (detachment, rounding and membrane permeation) are observed 36 hours after transfection with a high dosage of plasmid (500 ng) [39]. Control cells transfected with the same amount of an empty vector do not display features of cell death, suggesting that simply overdosing the cells with plasmid is not responsible for cell death [39]. Thus, we speculate that the reduced NF-κB activation from 370A observed by Fujimoto et al. [37] could be the result of induced cell death and that 370A in fact enhances NF-κB activation.
Our estimate of the time since fixation of 370A in a sample of 45 Chinese chromosomes is 10,740 years (Figure 4). This estimate involves several assumptions (see Materials and Methods) and should be interpreted with caution. Nevertheless the result suggests that 370A was likely at high frequency before the colonization of the Americas 10,500–14,000 years ago [41]–[43]. Thus, the high frequency of 370A in Native Americans (see Figure 1) is most likely due to positive selection prior to migrations from Asia to America.
In summary, we have demonstrated that the worldwide frequency distribution of 370A is highly unusual and that 370A was likely rising in frequency by positive selection in East Asia prior to 10,000 years ago. In addition, we have shown that the 370A allele results in enhanced NF-κB activation in vitro. What was the source of the selection pressure on 370A and what effect may 370A have on the phenotype? Since EDAR is involved in ectodermal development, 370A might be expected to affect teeth, hair, skin, nails and/or sweat glands. Fujimoto et al. [37] recently noted an association between 370A and hair thickness. Replication of this result is desirable since correction for population structure was inadequate: only a single SNP was used to correct for population structure. Nevertheless, the results of Fujimoto et al. [37] are suggestive, especially since East Asians have thicker hair than Europeans and Africans [38]. These observations lead us to question why thicker hair may have been advantageous in ancestral East Asian environments. Of course, thicker hair may not have been adaptive at all and may simply be the result of phenotypic hitchhiking: selection on 370A may have targeted a different phenotype (e.g. tooth morphology [16]) and hair thickness may have resulted as a by-product of this selection. Sexual selection also remains a possibility.
Regardless of the nature of the selective force, our results provide compelling evidence that positive selection has acted on the 370A allele in EDAR. In addition, our finding that 370A results in increased NF-κB activation suggests further lines for investigation: in particular, how does this increased NF-κB activation influence the expression of the target genes regulated by NF-κB? Such future studies will lead to a more complete understanding of the phenotypic effect of 370A, and will permit more explicit tests of hypotheses concerning the possible selection pressure(s) responsible for its rapid increase in frequency in East Asia.
Bryk J, Hardouin E, Pugach I, Hughes D, Strotmann R, Stoneking M, et al. (2008) Positive Selection in East Asians for an EDAR Allele that Enhances NF-κB Activation. PLoS ONE 3(5): e2209.
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