Ethical Guidance in Human Paleogenomics: New and Ongoing Perspectives.

Over the past two decades, the study of ancient genomes from Ancestral humans, or human paleogenomic research, has expanded rapidly in both scale and scope. Ethical discourse has subsequently emerged to address issues of social responsibility and scientific robusticity in conducting research. Here, we highlight and contextualize the primary sources of professional ethical guidance aimed at paleogenomic researchers. We describe the tension among existing guidelines, while addressing core issues such as consent, destructive research methods, and data access and management. Currently, there is a dissonance between guidelines that focus on scientific outcomes and those that hold scientists accountable to stakeholder communities,such as descendants. Thus, we provide additional tools to navigate the complexities of ancient DNA research while centering engagement with stakeholder communities in the scientific process.

Genetic research within Indigenous communities: Engagement opportunities and pathways forward.

PURPOSE: Against a historical backdrop of researchers who violated trust through lack of benefit sharing, transparency, and engagement, efforts are underway to develop better approaches for genetic and genomic research with Indigenous communities. To increase engagement, there is a need to understand factors that affect researcher and community collaborations. This study aimed to understand the barriers, challenges, and facilitators of Indigenous Peoples in the United States participating in genetic research. METHODS: We conducted 42 semistructured interviews with Tribal leaders, clinicians, researchers, policy makers, and Tribal research review board members across the United States to explore perceived risks, benefits, barriers, and facilitators of genetic research participation. RESULTS: Participants, identifying as Indigenous (88%) or non-Indigenous allies (12%), described their concerns, hesitancy, and fears about genetic research, as well as the roles of trust, transparency, and respect for culture in facilitating partnerships. Previous harms-such as sample and data misuse, stigmatization, or misrepresentation by researchers-revealed strategies for building trust to create more equitable and reciprocal research partnerships. CONCLUSION: Participants in this study offered strategies for increasing genetic research engagement. The pathway forward should foster transparent research policies and practices to facilitate informed research that supports the needs and priorities of participants, communities, and researchers.

Identification of Sociodemographic, Clinical, and Genetic Factors to Aid Alaska Native and American Indian People to Successfully Quit Smoking.

INTRODUCTION: Alaska Native and American Indian (ANAI) people have a smoking prevalence of 23%. Nicotine metabolite ratio (NMR) and genetic testing may enable tailored selection of tobacco cessation medication. AIMS AND METHODS: The purpose of this study was to evaluate the relative contributions of NMR, cessation medication, demographics, and tobacco use history to cessation. Participants were recruited into an observational cohort study consisting of a baseline visit prior to their quit date and 6-week follow-up. Demographic and tobacco use surveys and blood, urine, and breath samples were collected at each visit. Electronic health records were queried for cessation medications. NMR was categorized into slow or normal nicotine metabolism phenotypes (<0.31 and ≥ 0.31, respectively). The main outcome was cessation at 6 weeks. Analyses consisted of descriptive statistics, medication and phenotype concordance, and estimates of relative risk (RR) of quitting. RESULTS: We enrolled 151 ANAI adults who smoked cigarettes daily. Two-thirds had normal nicotine metabolism phenotype. Retrospective medication and phenotype concordance was 39%. The overall quit rate was 25%. No demographic factors or tobacco use history were associated with quit success. Varenicline and bupropion increased the likelihood of quitting (RR = 2.93 [1.42, 6.03] and RR = 2.52 [1.12, 5.64], respectively) compared to nicotine replacement therapy. Non-optimal medication and phenotype concordance decreased likelihood of quit success (RR = 0.44 [0.22, 0.91]) compared to optimal concordance. CONCLUSIONS: This exploratory study found associations between quit success and tobacco cessation medication as well as medication and phenotype concordance. Additional research is needed to assess use of NMR for treatment selection among ANAI people. IMPLICATIONS: These results broadly support additional community-engaged research to improve medication and phenotype concordance in tribal health settings. Such future research on implementing meditcation and phenotype concordance holds promise to improve expectations, quit success, and health outcomes amongst individuals attempting to quit smoking.

Fostering Responsible Research on Ancient DNA.

Anticipating and addressing the social implications of scientific work is a fundamental responsibility of all scientists. However, expectations for ethically sound practices can evolve over time as the implications of science come to be better understood. Contemporary researchers who work with ancient human remains, including those who conduct ancient DNA research, face precisely this challenge as it becomes clear that practices such as community engagement are needed to address the important social implications of this work. To foster and promote ethical engagement between researchers and communities, we offer five practical recommendations for ancient DNA researchers: (1) formally consult with communities; (2) address cultural and ethical considerations; (3) engage communities and support capacity building; (4) develop plans to report results and manage data; and (5) develop plans for long-term responsibility and stewardship. Ultimately, every member of a research team has an important role in fostering ethical research on ancient DNA.

Pharmacogenomics of Nicotine Metabolism: Novel CYP2A6 and CYP2B6 Genetic Variation Patterns in Alaska Native and American Indian Populations.

INTRODUCTION: Alaska Native and American Indian (AN/AI) populations have higher tobacco use prevalence than other ethnic/racial groups. Pharmacogenetic testing to tailor tobacco cessation treatment may improve cessation rates. This study characterized polymorphic variations among AN/AI people in genes associated with metabolism of nicotine and drugs used for tobacco cessation. METHODS: Recruitment of AN/AI individuals represented six subgroups, five geographic subgroups throughout Alaska and a subgroup comprised of AIs from the lower 48 states living in Alaska. We sequenced the CYP2A6 and CYP2B6 genes to identify known and novel gain, reduced, and loss-of-function alleles, including structural variation (eg, gene deletions, duplications, and hybridizations). RESULTS: Variant allele frequencies differed substantially between AN/AI subgroups. The gene deletion CYP2A6*4 and reduced function CYP2A6*9 alleles were found at high frequency in Northern/Western subgroups and in Lower 48/Interior subgroups, respectively. The reduced function CYP2B6*6 allele was observed in all subgroups and a novel, predicted reduced function CYP2B6 variant was found at relatively high frequency in the Southeastern subgroup. CONCLUSIONS: Diverse CYP2A6 and CYP2B6 variation among the subgroups highlight the need for comprehensive pharmacogenetic testing to guide tobacco cessation therapy for AN/AI populations. IMPLICATIONS: Nicotine metabolism is largely determined by CYP2A6 genotype, and variation in CYP2A6 activity has altered the treatment success in other populations. These findings suggest pharmacogenetic-guided smoking cessation drug treatment could provide benefit to this unique population seeking tobacco cessation therapy.

Weaving the Strands of Life (Iiná Bitl'ool): History of Genetic Research Involving Navajo People.

To date, some genetic studies offer medical benefits but lack a clear pathway to benefit for people from underrepresented backgrounds. Historically, Indigenous people, including the Diné (Navajo people), have raised concerns about the lack of benefits, misuse of DNA samples, lack of consultation, and ignoring of cultural and traditional ways of knowing. Shortly after the Navajo Nation Human Research Review Board was established in 1996, the Navajo Nation recognized growing concerns about genetic research, and in 2002 they established a moratorium on human genetic research studies. The moratorium effectively has protected their citizens from potential genetic research harms. Despite the placement of the moratorium, some genetic research studies have continued using blood and DNA samples from Navajo people. To understand the history of genetic research involving Navajo people, the authors conducted a literature review of genetic or genetics-related research publications that involved Navajo people, identifying 79 articles from the years 1926 to 2018. To their knowledge, no known literature review has comprehensively examined the history of genetic research in the Navajo community. This review divides the genetic research articles into the following general classifications: bacteria or virus genetics, blood and human leukocyte antigens, complex diseases, forensics, hereditary diseases, and population genetics and migration. The authors evaluated the methods reported in each article, described the number of Navajo individuals reported, recorded the academic and tribal approval statements, and noted whether the study considered Diné cultural values. Several studies focused on severe combined immunodeficiency disease, population history, neuropathy, albinism, and eye and skin disorders that affect Navajo people. The authors contextualize Diné ways of knowing related to genetics and health with Western scientific concepts to acknowledge the complex philosophy and belief system that guides Diné people and recognizes Indigenous science. They also encourage researchers to consider cultural perspectives and traditional knowledge that has the potential to create stronger conclusions and better-informed, ethical, and respectful science.

Quantitative evolutionary proteomics of seminal fluid from primates with different mating systems.

BACKGROUND: Genomic data from various organisms have been used to study how sexual selection has shaped genetic diversity in reproductive proteins, and in particular, to elucidate how mating systems may have influenced evolution at the molecular and phenotypic levels. However, large-scale proteomic data including protein identifications and abundances are only now entering the field of evolutionary and comparative genomics. Variation in both protein sequence and expression level may play important roles in the evolution of sexual traits and behaviors. RESULTS: Here, we broadly analyze the components of seminal fluid from primates with diverse mating systems ranging from monogamous to polygynous, and include genomics, proteomics, phylogenetic and quantitative characters into our framework. Our analyses show that seminal fluid proteins are undergoing rapid evolution and some of these quickly evolving proteins may be influenced by sexual selection. Through evolutionary analyses and protein abundance differences, we identified 84 genes whose evolutionary rates or expression levels were correlated with mating system and other sexual characters. We found that many proteins differ in abundance between monogamous and polygynous primate mating systems. Many of these proteins are enriched in the copulatory plug pathway, which suggests that post-zygotic selective barriers are important regardless of mating system type. CONCLUSIONS: This work is the first to comprehensively compare seminal fluid proteins between human and non-human primates using high-throughput proteomics. Our findings highlight the impact of mating system variation on seminal fluid protein evolution and abundance.

Novel CYP2A6 diplotypes identified through next-generation sequencing are associated with in-vitro and in-vivo nicotine metabolism.

OBJECTIVES: Smoking patterns and cessation rates vary widely across smokers and can be influenced by variation in rates of nicotine metabolism [i.e. cytochrome P450 2A6 (CYP2A6), enzyme activity]. There is high heritability of CYP2A6-mediated nicotine metabolism (60-80%) owing to known and unidentified genetic variation in the CYP2A6 gene. We aimed to identify and characterize additional genetic variants at the CYP2A6 gene locus. METHODS: A new CYP2A6-specific sequencing method was used to investigate genetic variation in CYP2A6. Novel variants were characterized in a White human liver bank that has been extensively phenotyped for CYP2A6. Linkage and haplotype structure for the novel single nucleotide polymorphisms (SNPs) were assessed. The association between novel five-SNP diplotypes and nicotine metabolism rate was investigated. RESULTS: Seven high-frequency (minor allele frequencies ≥6%) noncoding SNPs were identified as important contributors to CYP2A6 phenotypes in a White human liver bank (rs57837628, rs7260629, rs7259706, rs150298687 (also denoted rs4803381), rs56113850, rs28399453, and rs8192733), accounting for two times more variation in in-vitro CYP2A6 activity relative to the four established functional CYP2A6 variants that are frequently tested in Whites (CYP2A6*2, *4, *9, and *12). Two pairs of novel SNPs were in high linkage disequilibrium, allowing us to establish five-SNP diplotypes that were associated with CYP2A6 enzyme activity (rate of nicotine metabolism) in-vitro in the liver bank and in-vivo among smokers. CONCLUSION: The novel five-SNP diplotype may be useful to incorporate into CYP2A6 genotype models for personalized prediction of nicotine metabolism rate, cessation success, and response to pharmacotherapies.

Polymorphic Human Sulfotransferase 2A1 Mediates the Formation of 25-Hydroxyvitamin D3-3-O-Sulfate, a Major Circulating Vitamin D Metabolite in Humans.

Metabolism of 25-hydroxyvitamin D3 (25OHD3) plays a central role in regulating the biologic effects of vitamin D in the body. Although cytochrome P450-dependent hydroxylation of 25OHD3 has been extensively investigated, limited information is available on the conjugation of 25OHD3 In this study, we report that 25OHD3 is selectively conjugated to 25OHD3-3-O-sulfate by human sulfotransferase 2A1 (SULT2A1) and that the liver is a primary site of metabolite formation. At a low (50 nM) concentration of 25OHD3, 25OHD3-3-O-sulfate was the most abundant metabolite, with an intrinsic clearance approximately 8-fold higher than the next most efficient metabolic route. In addition, 25OHD3 sulfonation was not inducible by the potent human pregnane X receptor agonist, rifampicin. The 25OHD3 sulfonation rates in a bank of 258 different human liver cytosols were highly variable but correlated with the rates of dehydroepiandrosterone sulfonation. Further analysis revealed a significant association between a common single nucleotide variant within intron 1 of SULT2A1 (rs296361; minor allele frequency = 15% in whites) and liver cytosolic SULT2A1 content as well as 25OHD3-3-O-sulfate formation rate, suggesting that variation in the SULT2A1 gene contributes importantly to interindividual differences in vitamin D homeostasis. Finally, 25OHD3-3-O-sulfate exhibited high affinity for the vitamin D binding protein and was detectable in human plasma and bile but not in urine samples. Thus, circulating concentrations of 25OHD3-3-O-sulfate appear to be protected from rapid renal elimination, raising the possibility that the sulfate metabolite may serve as a reservoir of 25OHD3 in vivo, and contribute indirectly to the biologic effects of vitamin D.

Hepatic Abundance and Activity of Androgen- and Drug-Metabolizing Enzyme UGT2B17 Are Associated with Genotype, Age, and Sex.

The major objective of this study was to investigate the association of genetic and nongenetic factors with variability in protein abundance and in vitro activity of the androgen-metabolizing enzyme UGT2B17 in human liver microsomes (n = 455). UGT2B17 abundance was quantified by liquid chromatography-tandem mass spectrometry proteomics, and enzyme activity was determined by using testosterone and dihydrotestosterone as in vitro probe substrates. Genotyping or gene resequencing and mRNA expression were also evaluated. Multivariate analysis was used to test the association of UGT2B17 copy number variation, single nucleotide polymorphisms (SNPs), age, and sex with its mRNA expression, abundance, and activity. UGT2B17 gene copy number and SNPs (rs7436962, rs9996186, rs28374627, and rs4860305) were associated with gene expression, protein levels, and androgen glucuronidation rates in a gene dose-dependent manner. UGT2B17 protein (mean ± S.D. picomoles per milligram of microsomal protein) is sparsely expressed in children younger than 9 years (0.12 ± 0.24 years) but profoundly increases from age 9 years to adults (∼10-fold) with ∼2.6-fold greater abundance in males than in females (1.2 vs. 0.47). Association of androgen glucuronidation with UGT2B15 abundance was observed only in the low UGT2B17 expressers. These data can be used to predict variability in the metabolism of UGT2B17 substrates. Drug companies should include UGT2B17 in early phenotyping assays during drug discovery to avoid late clinical failures.

Responsible Research With Urban American Indians and Alaska Natives.

American Indian and Alaska Native (AI/AN) communities harbor understandable mistrust of research. Outside researchers have historically controlled processes, promulgating conclusions and recommended policies with virtually no input from the communities studied. Reservation-based communities can apply sovereignty rights conferred by the federal government to change this research trajectory. Many tribes now require review and approval before allowing research activities to occur, in part through the development of regulatory codes and oversight measures. Tribal oversight ensures that research is directed toward questions of importance to the community and that results are returned in ways that optimize problem solving. Unfortunately, tribal governance protections do not always extend to AI/ANs residing in urban environments. Although they represent the majority of AI/ANs, urban Indians face an ongoing struggle for visibility and access to health care. It is against this backdrop that urban Indians suffer disproportionate health problems. Improved efforts to ensure responsible research with urban Indian populations requires attention to community engagement, research oversight, and capacity building. We consider strategies to offset these limitations and develop a foundation for responsible research with urban Indians.

Predictors of Variation in CYP2A6 mRNA, Protein, and Enzyme Activity in a Human Liver Bank: Influence of Genetic and Nongenetic Factors.

Cytochrome P450 2A6 CYP2A6: metabolizes several clinically relevant substrates, including nicotine, the primary psychoactive component in cigarette smoke. Smokers vary widely in their rate of inactivation and clearance of nicotine, altering numerous smoking phenotypes. We aimed to characterize independent and shared impact of genetic and nongenetic sources of variation in CYP2A6 mRNA, protein, and enzyme activity in a human liver bank (n = 360). For the assessment of genetic factors, we quantified levels of CYP2A6, cytochrome P450 oxidoreductase (POR), and aldo-keto reductase 1D1 (AKR1D1) mRNA, and CYP2A6 and POR proteins. CYP2A6 enzyme activity was determined through measurement of cotinine formation from nicotine and 7-hydroxycoumarin formation from coumarin. Donor DNA was genotyped for CYP2A6, POR, and AKR1D1 genetic variants. Nongenetic factors assessed included gender, age, and liver disease. CYP2A6 phenotype measures were positively correlated to each other (r values ranging from 0.47-0.88, P < 0.001). Female donors exhibited higher CYP2A6 mRNA expression relative to males (P < 0.05). Donor age was weakly positively correlated with CYP2A6 protein (r = 0.12, P < 0.05) and activity (r = 0.20, P < 0.001). CYP2A6 reduced-function genotypes, but not POR or AKR1D1 genotypes, were associated with lower CYP2A6 protein (P < 0.001) and activity (P < 0.01). AKR1D1 mRNA was correlated with CYP2A6 mRNA (r = 0.57, P < 0.001), protein (r = 0.30, P < 0.001), and activity (r = 0.34, P < 0.001). POR protein was correlated with CYP2A6 activity (r = 0.45, P < 0.001). Through regression analyses, we accounted for 17% (P < 0.001), 37% (P < 0.001), and 77% (P < 0.001) of the variation in CYP2A6 mRNA, protein, and activity, respectively. Overall, several independent and shared sources of variation in CYP2A6 activity in vitro have been identified, which could translate to variable hepatic clearance of nicotine.

Genetic and Nongenetic Factors Associated with Protein Abundance of Flavin-Containing Monooxygenase 3 in Human Liver.

Hepatic flavin-containing mono-oxygenase 3 (FMO3) metabolizes a broad array of nucleophilic heteroatom (e.g., N or S)-containing xenobiotics (e.g., amphetamine, sulindac, benzydamine, ranitidine, tamoxifen, nicotine, and ethionamide), as well as endogenous compounds (e.g., catecholamine and trimethylamine). To predict the effect of genetic and nongenetic factors on the hepatic metabolism of FMO3 substrates, we quantified FMO3 protein abundance in human liver microsomes (HLMs; n = 445) by liquid chromatography-tandem mass chromatography proteomics. Genotyping/gene resequencing, mRNA expression, and functional activity (with benzydamine as probe substrate) of FMO3 were also evaluated. FMO3 abundance increased 2.2-fold (13.0 ± 11.4 pmol/mg protein vs. 28.0 ± 11.8 pmol/mg protein) from neonates to adults. After 6 years of age, no significant difference in FMO3 abundance was found between children and adults. Female donors exhibited modestly higher mRNA fragments per kilobase per million reads values (139.9 ± 76.9 vs. 105.1 ± 73.1; P < 0.001) and protein FMO3 abundance (26.7 ± 12.0 pmol/mg protein vs. 24.1 ± 12.1 pmol/mg protein; P < 0.05) compared with males. Six single nucleotide polymorphisms (SNPs), including rs2064074, rs28363536, rs2266782 (E158K), rs909530 (N285N), rs2266780 (E308G), and rs909531, were associated with significantly decreased protein abundance. FMO3 abundance in individuals homozygous and heterozygous for haplotype 3 (H3), representing variant alleles for all these SNPs (except rs2066534), were 50.8% (P < 0.001) and 79.5% (P < 0.01), respectively, of those with the reference homozygous haplotype (H1, representing wild-type). In summary, FMO3 protein abundance is significantly associated with age, gender, and genotype. These data are important in predicting FMO3-mediated heteroatom-oxidation of xenobiotics and endogenous biomolecules in the human liver.

Chaco Canyon Dig Unearths Ethical Concerns.

The field of paleogenomics (the study of ancient genomes) is rapidly advancing, with more robust methods of isolating ancient DNA and increasing access to next-generation DNA sequencing technology. As these studies progress, many important ethical issues have emerged that should be considered when ancient Native American remains, whom we refer to as ancestors, are used in research. We highlight a 2017 article by Kennett et al., "Archaeogenomic evidence reveals prehistoric matrilineal dynasty," that brings to light several ethical issues that should be addressed in paleogenomics research. The study helps elucidate the matrilineal relationships in ancient Chacoan society through ancient DNA analysis. However, we, as Indigenous researchers and allies, raise ethical concerns with the study's scientific conclusions that can be problematic for Native American communities: (1) the lack of tribal consultation, (2) the use of culturally insensitive descriptions, and (3) the potential impact on marginalized groups. Further, we explore the limitations of the Native American Graves Protection and Repatriation Act, which addresses repatriation but not research, because clear ethical guidelines have not been established for research involving Native American ancestors, especially those deemed "culturally unaffiliated." Multiple studies of "culturally unaffiliated" remains have been initiated recently, so it is imperative that researchers consider the ethical ramifications of paleogenomics research. Past research indiscretions have created a history of mistrust and exploitation in many Native American communities. To promote ethical engagement of Native American communities in research, we therefore suggest careful attention to ethical considerations, strong tribal consultation requirements, and greater collaborations among museums, federal agencies, researchers, scientific journals, and granting agencies.

Evolution of the egg: new findings and challenges.

The evolution of the egg is dynamic, and eggs have numerous species-specific properties across vertebrates and invertebrates. Interestingly, although the structure and function of the egg have remained relatively conserved over time, some constituents of the egg's extracellular barriers are undergoing rapid evolution. In this article, we review current ideas regarding sperm-egg interactions, discuss genetic approaches used to elucidate egg gene functions, and highlight the interesting differences that have evolved across taxa. We suggest that the rapid evolution of egg components and the mechanisms behind sperm-egg interactions are integrally connected, and delve in depth into each component of the egg's extracellular matrices. Finally, we discuss the promising future of reproductive research and how high-throughput genomics and proteomics have the potential to revolutionize the field and provide new evidence that will challenge previously held views about the fertilization process.

Detecting coevolution in mammalian sperm-egg fusion proteins.

Interactions between sperm and egg proteins can occur physically between gamete surface-binding proteins, and genetically between gamete proteins that work in complementary pathways in which they may not physically interact. Physically interacting sperm-egg proteins have been functionally identified in only a few species, and none have been verified within mammals. Candidate genes on both the sperm and egg surfaces exist, but gene deletion studies do not support functional interactions between these sperm-egg proteins; interacting sperm-egg proteins thus remain elusive. Cooperative gamete proteins undergo rapid evolution, and it is predicted that these sperm-egg proteins will also have correlated evolutionary rates due to compensatory changes on both the sperm and egg. To explore potential physical and genetic interactions in sperm-egg proteins, we sequenced four candidate genes from diverse primate species, and used regression and likelihood methods to test for signatures of coevolution between sperm-egg gene pairs. With both methods, we found that the egg protein CD9 coevolves with the sperm protein IZUMO1, suggesting a physical or genetic interaction occurs between them. With regression analysis, we found that CD9 and CRISP2 have correlated rates of evolution, and with likelihood analysis, that CD9 and CRISP1 have correlated rates. This suggests that the different tests may reflect different levels of interaction, be it physical or genetic. Coevolution tests thus provide an exploratory method for detecting potentially interacting sperm-egg protein pairs.

Cytochrome P450 Family 4F2 and 4F11 Haplotype Mapping and Association with Hepatic Gene Expression and Vitamin K Hydroxylation Activity.

This study evaluated the underlying mechanistic links between genetic variability in vitamin K metabolic pathway genes (CYP4F2 and CYP4F11) and phylloquinone hydroxylation activity using genotype- and haplotype-based approaches. Specifically, we characterized genetic variability in the CYP4F2/CYP4F11 locus and compared common single allele genotypes and common haplotypes as predictors of hepatic gene expression, enzyme abundance, and phylloquinone (VK1) ω-hydroxylation kinetics. We measured CYP4F2 and CYP4F11 mRNA levels, CYP4F2 and CYP4F11 protein abundances, and the VK1 concentration-dependent ω-hydroxylation rate in matched human liver nucleic acid and microsome samples, utilizing a novel in vitro population modeling approach. Results indicate that accounting for the CYP4F2*3 allele alone is sufficient to capture most of the genetic-derived variability in the observed phenotypes. Additionally, our findings highlight the important contribution that CYP4F11 makes toward vitamin K metabolism in the human liver.