Effects of cis and trans Genetic Ancestry on Gene Expression in African Americans

Variation in gene expression is a fundamental aspect of human phenotypic variation. Several recent studies have analyzed gene expression levels in populations of different continental ancestry and reported population differences at a large number of genes. However, these differences could largely be due to non-genetic (e.g., environmental) effects. Here, we analyze gene expression levels in African American cell lines, which differ from previously analyzed cell lines in that individuals from this population inherit variable proportions of two continental ancestries. We first relate gene expression levels in individual African Americans to their genome-wide proportion of European ancestry. The results provide strong evidence of a genetic contribution to expression differences between European and African populations, validating previous findings. Second, we infer local ancestry (0, 1, or 2 European chromosomes) at each location in the genome and investigate the effects of ancestry proximal to the expressed gene (cis) versus ancestry elsewhere in the genome (trans). Both effects are highly significant, and we estimate that 12±3% of all heritable variation in human gene expression is due to cis variants.     

Functional Characterization Improves Associations between Rare Non-Synonymous Variants in CHRNB4 and Smoking Behavior

Smoking is the leading cause of preventable death worldwide. Accordingly, effort has been devoted to determining the genetic variants that contribute to smoking risk. Genome-wide association studies have identified several variants in nicotinic acetylcholine receptor genes that contribute to nicotine dependence risk. We previously undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes and found that rare missense variants at conserved residues in CHRNB4 are associated with reduced risk of nicotine dependence among African Americans. We identified 10 low frequency (<5%) non-synonymous variants in CHRNB4 and investigated functional effects by co-expression with normal α3 or α4 subunits in human embryonic kidney cells. Voltage-clamp was used to obtain acetylcholine and nicotine concentration–response curves and qRT-PCR, western blots and cell-surface ELISAs were performed to assess expression levels. These results were used to functionally weight genetic variants in a gene-based association test. We find that there is a highly significant correlation between carrier status weighted by either acetylcholine EC₅₀ (β = −0.67, r² = 0.017, P = 2×10⁻⁴) or by response to low nicotine (β = −0.29, r² = 0.02, P = 6×10⁻⁵) when variants are expressed with the α3 subunit. In contrast, there is no significant association when carrier status is unweighted (β = −0.04, r² = 0.0009, P = 0.54). These results highlight the value of functional analysis of variants and the advantages to integrating such data into genetic studies. They also suggest that an increased sensitivity to low concentrations of nicotine is protective from the risk of developing nicotine dependence.     

Effects of NR1H3 Genetic Variation on the Expression of Liver X Receptor α and the Progression of Alzheimer's Disease

Alzheimer''s disease (AD) has been postulated to involve defects in the clearance of amyloid-β (Aβ). Activation of liver X receptor α (LXRα) increases the expression of apolipoprotein E (ApoE) as well as cholesterol transporters ABCA1 and ABCG1, leading to augmented clearance of Aβ. We have previously shown that the C allele of rs7120118 in the NR1H3 gene encoding LXRα reduces the risk of AD. Here, we wanted to assess whether the rs7120118 variation affects the progression of AD and modulates the expression of NR1H3 and its downstream targets APOE, ABCA1 and ABCG1.We utilized tissue samples from the inferior temporal cortex of 87 subjects, which were subdivided according to Braak staging into mild, moderate and severe AD groups on the basis of AD-related neurofibrillary pathology. APOE ε4 allele increased soluble Aβ42 levels in the tissue samples in a dose-dependent manner, but did not affect the expression status of APOE. In contrast, the CC genotype of rs7120118 was underrepresented in the severe group, although this result did not reach statistical significance. Also, patients with the CC genotype of rs7120118 showed significantly decreased soluble Aβ42 levels as compared to the patients with TT genotype. Although the severity of AD did not affect NR1H3 expression, the mRNA levels of NR1H3 among the patients with CT genotype of rs7120118 were significantly increased as compared to the patients with TT genotype. These results suggest that genetic variation in NR1H3 modulates the expression of LXRα and the levels of soluble Aβ42.     

Multiple Loci Associated with Renal Function in African Americans

The incidence of chronic kidney disease varies by ethnic group in the USA, with African Americans displaying a two-fold higher rate than European Americans. One of the two defining variables underlying staging of chronic kidney disease is the glomerular filtration rate. Meta-analysis in individuals of European ancestry has identified 23 genetic loci associated with the estimated glomerular filtration rate (eGFR). We conducted a follow-up study of these 23 genetic loci using a population-based sample of 1,018 unrelated admixed African Americans. We included in our follow-up study two variants in APOL1 associated with end-stage kidney disease discovered by admixture mapping in admixed African Americans. To address confounding due to admixture, we estimated local ancestry at each marker and global ancestry. We performed regression analysis stratified by local ancestry and combined the resulting regression estimates across ancestry strata using an inverse variance-weighted fixed effects model. We found that 11 of the 24 loci were significantly associated with eGFR in our sample. The effect size estimates were not significantly different between the subgroups of individuals with two copies of African ancestry vs. two copies of European ancestry for any of the 11 loci. In contrast, allele frequencies were significantly different at 10 of the 11 loci. Collectively, the 11 loci, including four secondary signals revealed by conditional analyses, explained 14.2% of the phenotypic variance in eGFR, in contrast to the 1.4% explained by the 24 loci in individuals of European ancestry. Our findings provide insight into the genetic basis of variation in renal function among admixed African Americans.     

Associations of Variants in CHRNA5/A3/B4 Gene Cluster with Smoking Behaviors in a Korean Population

Multiple genome-wide and targeted association studies reveal a significant association of variants in the CHRNA5-CHRNA3-CHRNB4 (CHRNA5/A3/B4) gene cluster on chromosome 15 with nicotine dependence. The subjects examined in most of these studies had a European origin. However, considering the distinct linkage disequilibrium patterns in European and other ethnic populations, it would be of tremendous interest to determine whether such associations could be replicated in populations of other ethnicities, such as Asians. In this study, we performed comprehensive association and interaction analyses for 32 single-nucleotide polymorphisms (SNPs) in CHRNA5/A3/B4 with smoking initiation (SI), smoking quantity (SQ), and smoking cessation (SC) in a Korean sample (N = 8,842). We found nominally significant associations of 7 SNPs with at least one smoking-related phenotype in the total sample (SI: P = 0.015∼0.023; SQ: P = 0.008∼0.028; SC: P = 0.018∼0.047) and the male sample (SI: P = 0.001∼0.023; SQ: P = 0.001∼0.046; SC: P = 0.01). A spectrum of haplotypes formed by three consecutive SNPs located between rs16969948 in CHRNA5 and rs6495316 in the intergenic region downstream from the 5′ end of CHRNB4 was associated with these three smoking-related phenotypes in both the total and the male sample. Notably, associations of these variants and haplotypes with SC appear to be much weaker than those with SI and SQ. In addition, we performed an interaction analysis of SNPs within the cluster using the generalized multifactor dimensionality reduction method and found a significant interaction of SNPs rs7163730 in LOC123688, rs6495308 in CHRNA3, and rs7166158, rs8043123, and rs11072793 in the intergenic region downstream from the 5′ end of CHRNB4 to be influencing SI in the male sample. Considering that fewer than 5% of the female participants were smokers, we did not perform any analysis on female subjects specifically. Together, our detected associations of variants in the CHRNA5/A3/B4 cluster with SI, SQ, and SC in the Korean smoker samples provide strong evidence for the contribution of this cluster to the etiology of SI, ND, and SC in this Asian population.     

Replication and fine mapping of asthma-associated loci in individuals of African ancestry

Asthma originates from genetic and environmental factors with about half the risk of disease attributable to heritable causes. Genome-wide association studies, mostly in populations of European ancestry, have identified numerous asthma-associated single nucleotide polymorphisms (SNPs). Studies in populations with diverse ancestries allow both for identification of robust associations that replicate across ethnic groups and for improved resolution of associated loci due to different patterns of linkage disequilibrium between ethnic groups. Here we report on an analysis of 745 African-American subjects with asthma and 3,238 African-American control subjects from the Candidate Gene Association Resource (CARe) Consortium, including analysis of SNPs imputed using 1,000 Genomes reference panels and adjustment for local ancestry. We show strong evidence that variation near RAD50/IL13, implicated in studies of European ancestry individuals, replicates in individuals largely of African ancestry. Fine mapping in African ancestry populations also refined the variants of interest for this association. We also provide strong or nominal evidence of replication at loci near ORMDL3/GSDMB, IL1RL1/IL18R1, and 10p14, all previously associated with asthma in European or Japanese populations, but not at the PYHIN1 locus previously reported in studies of African-American samples. These results improve the understanding of asthma genetics and further demonstrate the utility of genetic studies in populations other than those of largely European ancestry.     

Multiple cholinergic nicotinic receptor genes affect nicotine dependence risk in African and European Americans

Several independent studies show that the chromosome 15q25.1 region, which contains the CHRNA5–CHRNA3–CHRNB4 gene cluster, harbors variants strongly associated with nicotine dependence, other smoking behaviors, lung cancer and chronic obstructive pulmonary disease. We investigated whether variants in other cholinergic nicotinic receptor subunit (CHRN) genes affect the risk of nicotine dependence in a new sample of African Americans (AAs) (N = 710). We also analyzed this AA sample together with a European American (EA) sample (N = 2062, 1608 of which have been previously studied), allowing for differing effects in the two populations. Cases are current nicotine‐dependent smokers and controls are non‐dependent smokers. Variants in or near CHRND–CHRNG, CHRNA7 and CHRNA10 show modest association with nicotine dependence risk in the AA sample. In addition, CHRNA4, CHRNB3–CHRNA6 and CHRNB1 show association in at least one population. CHRNG and CHRNA4 harbor single nucleotide polymorphisms (SNPs) that have opposite directions of effect in the two populations. In each of the population samples, these loci substantially increase the trait variation explained, although no loci meet Bonferroni‐corrected significance in the AA sample alone. The trait variation explained by three key associated SNPs in CHRNA5–CHRNA3–CHRNB4 is 1.9% in EAs and also 1.9% in AAs; this increases to 4.5% in EAs and 7.3% in AAs when we add six variants representing associations at other CHRN genes. Multiple nicotinic receptor subunit genes outside chromosome 15q25 are likely to be important in the biological processes and development of nicotine dependence, and some of these risks may be shared across diverse populations.     

Omental adipose tissue gene expression,gene variants,branched-chain amino acids,and their relationship with metabolic syndrome and insulin resistance in humans

Obesity is a complex disorder caused by several factors. Thus, the aim of the present study was to assess whether the expression of genes in the omental white adipose tissue (AT) of subjects with insulin resistance (IR) or metabolic syndrome (MetS) is associated with an elevation in serum branched-chain amino acids (BCAAs) and whether this response depends on specific genetic variants. Serum BCAA concentration, the adipocyte area, and gene variants of PPARγ, ABCA1, FTO, TCF7L2, GFOD2,BCAT2, and BCKDH were determined in 115 Mexican subjects. The gene expression in the AT and adipocytes of BCAT, BCKDH E1α, C/EBPα, PPARγ2, SREBP-1, PPARα, UCP1, leptin receptor, leptin, adiponectin, and TNFα was measured in 51 subjects. Subjects with IR showed higher values for the BMI, HOMA-IR, and adipocyte area and higher levels of serum glucose, insulin, leptin, and C-reactive protein, as well as an elevation of the AT gene expression of SREBP-1, leptin, and TNFα and a significant reduction in the expression of adiponectin, BCAT2, and BCKDH E1α, compared with non-IR subjects. The presence of MetS was associated with higher HOMA-IR as well as higher serum BCAA concentrations. Subjects with the genetic variants for BCAT2 and BCKDH E1 α showed a lower serum BCAA concentration, and those with the ABCA1 and FTO gene variant showed higher levels of insulin and HOMA-IR than non-IR subjects. AT dysfunction is the result of a combination of the presence of some genetic variants, altered AT gene expression, the presence of MetS risk factors, IR, and serum BCAA concentrations.     

Genetic Differences between the Determinants of Lipid Profile Phenotypes in African and European Americans: The Jackson Heart Study

Genome-wide association analysis in populations of European descent has recently found more than a hundred genetic variants affecting risk for common disease. An open question, however, is how relevant the variants discovered in Europeans are to other populations. To address this problem for cardiovascular phenotypes, we studied a cohort of 4,464 African Americans from the Jackson Heart Study (JHS), in whom we genotyped both a panel of 12 recently discovered genetic variants known to predict lipid profile levels in Europeans and a panel of up to 1,447 ancestry informative markers allowing us to determine the African ancestry proportion of each individual at each position in the genome. Focusing on lipid profiles—HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), and triglycerides (TG)—we identified the lipoprotein lipase (LPL) locus as harboring variants that account for interethnic variation in HDL-C and TG. In particular, we identified a novel common variant within LPL that is strongly associated with TG (p=2.7×10⁻⁶) and explains nearly 1% of the variability in this phenotype, the most of any variant in African Americans to date. Strikingly, the extensively studied “gain-of-function” S447X mutation at LPL, which has been hypothesized to be the major determinant of the LPL-TG genetic association and is in trials for human gene therapy, has a significantly diminished strength of biological effect when it is found on a background of African rather than European ancestry. These results suggest that there are other, yet undiscovered variants at the locus that are truly causal (and are in linkage disequilibrium with S447X) or that work synergistically with S447X to modulate TG levels. Finally, we find systematically lower effect sizes for the 12 risk variants discovered in European populations on the African local ancestry background in JHS, highlighting the need for caution in the use of genetic variants for risk assessment across different populations.     

Inhibin Alpha-Subunit (INHA) Expression in Adrenocortical Cancer Is Linked to Genetic and Epigenetic INHA Promoter Variation

Adrenocortical carcinoma (ACC) is a rare, but highly malignant tumor of unknown origin. Inhibin α-subunit (Inha) knockout mice develop ACCs following gonadectomy. In man, INHA expression varies widely within ACC tissues and its circulating peptide inhibin pro-αC has been described as a novel tumor marker for ACC. We investigated whether genetic and epigenetic changes of the INHA gene in human ACC cause loss or variation of INHA expression. To this end, analyses of INHA sequence, promoter methylation and mRNA expression were performed in human adrenocortical tissues. Serum inhibin pro-αC levels were also measured in ACC patients. INHA genetic analysis in 37 unique ACCs revealed 10 novel, heterozygous rare variants. Of the 3 coding bases affected, one variant was synonymous and two were missense variants: S72F and S184F. The minor allele of rs11893842 at −124 bp was observed at a low frequency (24%) in ACC samples and was associated with decreased INHA mRNA levels: 4.7±1.9 arbitrary units for AA, compared to 26±11 for AG/GG genotypes (P = 0.034). The methylation of four proximal INHA promoter CpGs was aberrantly increased in five ACCs (47.7±3.9%), compared to normal adrenals (18.4±0.6%, P = 0.0052), whereas the other 14 ACCs studied showed diminished promoter methylation (9.8±1.1%, P = 0.020). CpG methylation was inversely correlated to INHA mRNA levels in ACCs (r = −0.701, p = 0.0036), but not associated with serum inhibin pro-αC levels. In conclusion, aberrant methylation and common genetic variation in the INHA promoter occur in human ACCs and are associated with decreased INHA expression.     

Gene-Based Sequencing Identifies Lipid-Influencing Variants with Ethnicity-Specific Effects in African Americans

Although a considerable proportion of serum lipids loci identified in European ancestry individuals (EA) replicate in African Americans (AA), interethnic differences in the distribution of serum lipids suggest that some genetic determinants differ by ethnicity. We conducted a comprehensive evaluation of five lipid candidate genes to identify variants with ethnicity-specific effects. We sequenced ABCA1, LCAT, LPL, PON1, and SERPINE1 in 48 AA individuals with extreme serum lipid concentrations (high HDLC/low TG or low HDLC/high TG). Identified variants were genotyped in the full population-based sample of AA (n = 1694) and tested for an association with serum lipids. rs328 (LPL) and correlated variants were associated with higher HDLC and lower TG. Interestingly, a stronger effect was observed on a “European” vs. “African” genetic background at this locus. To investigate this effect, we evaluated the region among West Africans (WA). For TG, the effect size among WA was the same in AA with only African local ancestry (2–3% lower TG), while the larger association among AA with local European ancestry matched previous reports in EA (10%). For HDLC, there was no association with rs328 in AA with only African local ancestry or in WA, while the association among AA with European local ancestry was much greater than what has been observed for EA (15 vs. ∼5 mg/dl), suggesting an interaction with an environmental or genetic factor that differs by ethnicity. Beyond this ancestry effect, the importance of African ancestry-focused, sequence-based work was also highlighted by serum lipid associations of variants that were in higher frequency (or present only) among those of African ancestry. By beginning our study with the sequence variation present in AA individuals, investigating local ancestry effects, and seeking replication in WA, we were able to comprehensively evaluate the role of a set of candidate genes in serum lipids in AA.     

A Human-Specific α7-Nicotinic Acetylcholine Receptor Gene in Human Leukocytes: Identification,Regulation and the Consequences of CHRFAM7A Expression

The human genome contains a variant form of the α7-nicotinic acetylcholine receptor (α7nAChR) gene that is uniquely human. This CHRFAM7A gene arose during human speciation and recent data suggests that its expression alters ligand tropism of the normally homopentameric human α7-AChR ligand-gated cell surface ion channel that is found on the surface of many different cell types. To understand its possible significance in regulating inflammation in humans, we investigated its expression in normal human leukocytes and leukocyte cell lines, compared CHRFAM7A expression to that of the CHRNA7 gene, mapped its promoter and characterized the effects of stable CHRFAM7A overexpression. We report here that CHRFAM7A is highly expressed in human leukocytes but that the levels of both CHRFAM7A and CHRNA7 mRNAs were independent and varied widely. To this end, mapping of the CHRFAM7A promoter in its 5′-untranslated region (UTR) identified a unique 1-kb sequence that independently regulates CHRFAM7A gene expression. Because overexpression of CHRFAM7A in THP1 cells altered the cell phenotype and modified the expression of genes associated with focal adhesion (for example, FAK, P13K, Akt, rho, GEF, Elk1, CycD), leukocyte transepithelial migration (Nox, ITG, MMPs, PKC) and cancer (kit, kitL, ras, cFos cyclinD1, Frizzled and GPCR), we conclude that CHRFAM7A is biologically active. Most surprisingly however, stable CHRFAM7A overexpression in THP1 cells upregulated CHRNA7, which, in turn, led to increased binding of the specific α7nAChR ligand, bungarotoxin, on the THP1 cell surface. Taken together, these data confirm the close association between CHRFAM7A and CHRNA7 expression, establish a biological consequence to CHRFAM7A expression in human leukocytes and support the possibility that this human-specific gene might contribute to, and/or gauge, a human-specific response to inflammation.     

Inherited hemoglobin disorders in an Afro-Amazonian community: Saracura

The most common hemoglobinopathies, viz, hemoglobins S and C, and α- and β-thalassemias, were investigated through the molecular screening of 116 subjects from the community of Saracura, comprising fugitive African slaves from farms of the municipality of Santarém, in the west of Pará State, Brazilian Amazon. The observed frequency of the HBB*S gene (0.9%) was significantly lower than that encountered in other Afro-derived communities in the region. Concomitantly, the absence of the HBB*C allele has been reported for most of the Afro-Amazonian communities thus far studied. As remnant populations of quilombos are generally small, the heterogeneous distribution of HBB*S and HBB*C alleles among them is probably due to genetic drift and/or founder effect. The observed frequency of 3.7 kb deletion in Saracura (8.5%) was consistent with the African origin of the population, with a certain degree of local differentiation and admixture with individuals of Caucasian ancestry, placed in evidence by the occurrence of - -(MED) deletion (1.2%), a common mutation in Mediterranean regions. As regards β-thalassemia, among the seven different mutations found in Saracura, three β^o and two β⁺ mutations were of Mediterranean origin, and two β⁺ of African. Thus, only 28% of the local β-thalassemia mutations found in Saracura were of African origin.     

Multiple insulin degrading enzyme variants alter in vitro reporter gene expression

The insulin degrading enzyme (IDE) variant, v311 (rs6583817), is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma β-amyloid (Aβ), decreased risk for Alzheimer''s disease (AD) and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA) and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957), for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2) successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p = 0.04) but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5′ to the promoter (1.18 fold-change, p = 0.006) and 3′ to the reporter gene (1.29 fold change, p = 0.003), an effect contributed to by four variants (v10, v315, v154 and v311). Since IDE mediates Aβ degradation, variants that regulate IDE expression could represent good therapeutic targets for AD.     

DRD2/CHRNA5 Interaction on Prefrontal Biology and Physiology during Working Memory

Background

Prefrontal behavior and activity in humans are heritable. Studies in animals demonstrate an interaction between dopamine D2 receptors and nicotinic acetylcholine receptors on prefrontal behavior but evidence in humans is weak. Therefore, we hypothesize that genetic variation regulating dopamine D2 and nicotinic acetylcholine receptor signaling impact prefrontal cortex activity and related cognition. To test this hypothesis in humans, we explored the interaction between functional genetic variants in the D2 receptor gene (DRD2, rs1076560) and in the nicotinic receptor α5 gene (CHRNA5, rs16969968) on both dorsolateral prefrontal cortex mediated behavior and physiology during working memory and on prefrontal gray matter volume.

Methods

A large sample of healthy subjects was compared for genotypic differences for DRD2 rs1076560 (G>T) and CHNRA5 rs16969968 (G>A) on prefrontal phenotypes, including cognitive performance at the N-Back task, prefrontal physiology with BOLD fMRI during performance of the 2-Back working memory task, and prefrontal morphometry with structural MRI.

Results

We found that DRD2 rs1076560 and CHNRA5 rs16969968 interact to modulate cognitive function, prefrontal physiology during working memory, and prefrontal gray matter volume. More specifically, CHRNA5-AA/DRD2-GT subjects had greater behavioral performance, more efficient prefrontal cortex activity at 2Back working memory task, and greater prefrontal gray matter volume than the other genotype groups.

Conclusions

The present data extend previous studies in animals and enhance our understanding of dopamine and acetylcholine signaling in the human prefrontal cortex, demonstrating interactions elicited by working memory that are modulated by genetic variants in DRD2 and CHRNA5.