Cardiovascular disease (CVD) events due to atherosclerosis cause one-third of worldwide deaths and risk factors include physical inactivity, age, dyslipidemia, hypertension, diabetes, obesity, smoking, and red meat consumption. However, ∼15% of first-time events occur without such factors. In contrast, coronary events are extremely rare even in closely related chimpanzees in captivity, despite human-like CVD–risk-prone blood lipid profiles, hypertension, and mild atherosclerosis. Similarly, red meat-associated enhancement of CVD event risk does not seem to occur in other carnivorous mammals. Thus, heightened CVD risk may be intrinsic to humans, and genetic changes during our evolution need consideration. Humans exhibit a species-specific deficiency of the sialic acid N-glycolylneuraminic acid (Neu5Gc), due to pseudogenization of cytidine monophosphate-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH), which occurred in hominin ancestors ∼2 to 3 Mya. Ldlr−/− mice with human-like Cmah deficiency fed a sialic acids (Sias)-free high-fat diet (HFD) showed ∼1.9-fold increased atherogenesis over Cmah wild-type Ldlr−/− mice, associated with elevated macrophage cytokine expression and enhanced hyperglycemia. Human consumption of Neu5Gc (from red meat) acts as a “xeno-autoantigen” via metabolic incorporation into endogenous glycoconjugates, as interactions with circulating anti-Neu5Gc “xeno-autoantibodies” potentiate chronic inflammation (“xenosialitis”). Cmah−/−Ldlr−/− mice immunized with Neu5Gc-bearing antigens to generate human-like anti-Neu5Gc antibodies suffered a ∼2.4-fold increased atherosclerosis on a Neu5Gc-rich HFD, compared with Neu5Ac-rich or Sias-free HFD. Lesions in Neu5Gc-immunized and Neu5Gc-rich HFD-fed Cmah−/−Ldlr−/− mice were more advanced but unexplained by lipoprotein or glucose changes. Human evolutionary loss of CMAH likely contributes to atherosclerosis predisposition via multiple intrinsic and extrinsic mechanisms, and future studies could consider this more human-like model.

Compared with our closest living evolutionary cousins, humans appear unusually prone to develop carcinomas (cancers arising from epithelia). The SIGLEC12 gene, which encodes the Siglec‐XII protein expressed on epithelial cells, has several uniquely human features: a fixed homozygous missense mutation inactivating its natural ligand recognition property; a polymorphic frameshift mutation eliminating full‐length protein expression in ~60%–70% of worldwide human populations; and, genomic features suggesting a negative selective sweep favoring the pseudogene state. Despite the loss of canonical sialic acid binding, Siglec‐XII still recruits Shp2 and accelerates tumor growth in a mouse model. We hypothesized that dysfunctional Siglec‐XII facilitates human carcinoma progression, correlating with known tumorigenic signatures of Shp2‐dependent cancers. Immunohistochemistry was used to detect Siglec‐XII expression on tissue microarrays. PC‐3 prostate cancer cells were transfected with Siglec‐XII and transcription of genes enriched with Siglec‐XII was determined. Genomic SIGLEC12 status was determined for four different cancer cohorts. Finally, a dot blot analysis of human urinary epithelial cells was established to determine the Siglec‐XII expressors versus non‐expressors. Forced expression in a SIGLEC12 null carcinoma cell line enriched transcription of genes associated with cancer progression. While Siglec‐XII was detected as expected in ~30%–40% of normal epithelia, ~80% of advanced carcinomas showed strong expression. Notably, >80% of late‐stage colorectal cancers had a functional SIGLEC12 allele, correlating with overall increased mortality. Thus, advanced carcinomas are much more likely to occur in individuals whose genomes have an intact SIGLEC12 gene, likely because the encoded Siglec‐XII protein recruits Shp2‐related oncogenic pathways. The finding has prognostic, diagnostic, and therapeutic implications.

Human-specific pseudogenization of the CMAH gene eliminated the mammalian sialic acid (Sia) Neu5Gc (generating an excess of its precursor Neu5Ac), thus changing ubiquitous cell surface “self-associated molecular patterns” (SAMPs) that modulate innate immunity via engagement of CD33-related-Siglec receptors. The Alu-fusion-mediated loss-of-function of CMAH fixed ∼2-3 million years ago (Mya), possibly contributing to the origins of the genus Homo. The mutation likely altered human SAMPs, triggering multiple events, including emergence of human-adapted pathogens with strong preference for Neu5Ac recognition and/or presenting Neu5Ac-containing molecular mimics of human glycans, which can suppress immune responses via CD33-related-Siglec engagement. Human-specific alterations reported in some genes encoding Sia-sensing proteins suggested a “hotspot” in hominin evolution. The availability of more hominid genomes including those of two extinct hominins now allows full reanalysis and evolutionary timing. Functional changes occur in 8/13 members of the human genomic cluster encoding CD33-related-Siglecs, all predating the human common ancestor. Comparisons with great ape genomes indicate that these changes are unique to hominins. We found no evidence for strong selection after the Human-Neanderthal/Denisovan common ancestor, and these extinct hominin genomes include almost all major changes found in humans, indicating that these changes in hominin sialobiology predate the Neanderthal-human divergence ∼0.6Mya. Multiple changes in this genomic cluster may also explain human-specific expression of CD33rSiglecs in unexpected locations such as amnion, placental trophoblast, pancreatic islets, ovarian fibroblasts, microglia, NK cells and epithelia. Taken together, our data suggest that innate immune interactions with pathogens markedly altered hominin Siglec biology between 0.6-2 Mya, potentially affecting human evolution.

Cardiovascular disease (CVD) events due to atherosclerosis are very common in humans, but rarely occur spontaneously in other mammals, absent experimental manipulation. All humans exhibit a species-specific deficiency of the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), due to pseudogenization of the CMP-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) gene, which occurred in hominin ancestors about 2-3 million years ago. Human-like Cmah -/- mice that express only the precursor sialic acid Neu5Ac are more prone to insulin resistance and have more reactive macrophages and T cells. Human dietary consumption of Neu5Gc (primarily from red meat), can acts as a foreign “xenoantigen” in humans that gets metabolically incorporated into endogenous glycoproteins. Humans with circulating anti-Neu5Gc “xeno-autoantibodies” can thus potentially develop local chronic inflammation or “xenosialitis” at sites of Neu5Gc accumulation such as endothelial cells and in atherosclerotic plaques. In this study we set out to test if human CMAH deficiency contributes to CVD via multiple intrinsic and extrinsic mechanisms. Cmah -/- Ldlr -/- mice had increased atherogenesis on a Neu5Gc-free high fat diet (HFD), compared to Cmah +/+ Ldlr -/- mice. This was not associated with cytokine levels in plasma, but increased cytokine expression was seen in Cmah -/- Ldlr -/- macrophages in comparison to Cmah +/+ Ldlr -/- . The baseline relative hyperglycemia of the Cmah -/- Ldlr -/- mice was also enhanced on a Neu5Gc-free HFD. When such mice were immunized to develop human-like levels of anti-Neu5Gc antibodies, they had a 2.5-fold increase in atherosclerosis on a Neu5Gc-rich HFD compared to control Neu5Ac-rich or sialic acid-free HFD feeding. Drastically advanced lesions with increased necrotic core areas and infiltration of macrophages and T-cells accompanied increases in atherosclerotic area and lesion volume. None of these differences were explained by changes in lipoprotein profiles or insulin sensing. Human evolutionary loss of CMAH likely contributes to atherosclerosis propensity, via both intrinsic mechanisms such as amplified chronic inflammatory response and hyperglycemia; and extrinsic mechanisms such as red meat-derived Neu5Gc-induced xenosialitis.

Human Siglec‐XII (a receptor encoded by the SIGLEC12 gene) is expressed in tissue macrophages and some epithelial cell surfaces. Humans (but not the closely related “great apes”) harbor a universally fixed missense mutation that eliminated the sialic acid binding property of this protein, a canonical functional feature of all other human Siglecs. Population analysis of the SIGLEC12 locus identified a polymorphic frameshift mutation, which leads to truncation of the Siglec‐XII polypeptide, and loss of expression. The homozygous null state of this mutation is present in all human populations with an average frequency of ~40%, and the locus was independently identified as undergoing an unexplained “negative selective sweep”, apparently favoring the null state. In the current study, immunohistochemistry using a monoclonal antibody to Siglec‐XII showed that while some epithelia in ~35% of normal human tissue samples from human autopsies express Siglec‐XII on epithelial cells (approximately the expected rate based on the frequency of the null state), high expression was observed in about 80% of samples from advanced carcinomas. Genomic studies on population cohorts with a low frequency of carcinomas (Seventh‐day Adventists) and in early stage prostate cancers diagnosed clinically by PSA expression did not show any correlation of disease risk or early‐stage progression with the SIGLEC12 frameshift mutation. In contrast, analysis of two advanced colorectal cancer cohorts (TRIBE and FIRE3) demonstrated that more than 80% of the patients had wild‐type SIGLEC12. Moreover, in one of the colorectal cancer cohorts (FIRE3), the presence of wild‐type SIGLEC12 was correlated with significantly decreased survival. Meanwhile, gene expression data comparing a tumor cell line transfected with a human SIGLEC12 cDNA with sham‐transfected cells shows upregulation of pathways associated with cancer progression. Taken together, all of these evolutionary clues and experimental findings suggest that while SIGLEC12 likely lost its function in the immune system of humans prior to the origin of our species, its persistence in some humans could be associated with the pathobiology of late stage carcinomas. In this regard, we have taken advantage of the expression of wild type Siglec‐XII on urinary epithelial cells, to develop a simple urine test to check for the expression status of SIGLEC12. This approach can be combined with future genomic analyses and signaling studies, to explore this unusual human specific evolution of an apparent evolutionary liability.Support or Funding InformationThe G. Harold & Leila Y. Mathers Charitable Foundation grantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.