Association of genetic variants in complement factor H and factor H-related genes with systemic lupus erythematosus susceptibility

Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, P _meta = 6.6×10⁻⁸, OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, P _meta = 2.9×10⁻⁷, OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ∼146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (P _meta = 3.2×10⁻⁷, OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (P _meta = 3.5×10⁻⁴, OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE.     

Comprehensive genotyping in two homogeneous Graves' disease samples reveals major and novel HLA association alleles

Background

Graves'' disease (GD) is the leading cause of hyperthyroidism and thyroid eye disease inherited as a complex trait. Although geoepidemiology studies showed relatively higher prevalence of GD in Asians than in Caucasians, previous genetic studies were contradictory concerning whether and/or which human leukocyte antigen (HLA) alleles are associated with GD in Asians.

Methodology/Principal Findings

We conducted a case-control association study (499 unrelated GD cases and 504 controls) and a replication in an independent family sample (419 GD individuals and their 282 relatives in 165 families). To minimize genetic and phenotypic heterogeneity, we included only ethnic Chinese Han population in Taiwan and excluded subjects with hypothyroidism. We performed direct and comprehensive genotyping of six classical HLA loci (HLA-A, -B, -C, -DPB1, -DQB1 and -DRB1) to 4-digit resolution. Combining the data of two sample populations, we found that B*46:01 (odds ratio under dominant model [OR]  = 1.33, Bonferroni corrected combined P [P_Bc]  = 1.17×10⁻²), DPB1*05:01 (OR  = 2.34, P_Bc = 2.58×10⁻¹⁰), DQB1*03:02 (OR  = 0.62, P_Bc  = 1.97×10⁻²), DRB1*15:01 (OR  = 1.68, P_Bc = 1.22×10⁻²) and DRB1*16:02 (OR  = 2.63, P_Bc  = 1.46×10⁻⁵) were associated with GD. HLA-DPB1*05:01 is the major gene of GD in our population and singly accounts for 48.4% of population-attributable risk.

Conclusions/Significance

These GD-associated alleles we identified in ethnic Chinese Hans, and those identified in other Asian studies, are totally distinct from the known associated alleles in Caucasians. Identification of population-specific association alleles is the critical first step for individualized medicine. Furthermore, comparison between different susceptibility/protective alleles across populations could facilitate generation of novel hypothesis about GD pathophysiology and indicate a new direction for future investigation.     

Evidence for more than one Parkinson's disease-associated variant within the HLA region

Parkinson''s disease (PD) was recently found to be associated with HLA in a genome-wide association study (GWAS). Follow-up GWAS''s replicated the PD-HLA association but their top hits differ. Do the different hits tag the same locus or is there more than one PD-associated variant within HLA? We show that the top GWAS hits are not correlated with each other (0.00≤r²≤0.15). Using our GWAS (2000 cases, 1986 controls) we conducted step-wise conditional analysis on 107 SNPs with P<10⁻³ for PD-association; 103 dropped-out, four remained significant. Each SNP, when conditioned on the other three, yielded P_SNP1 = 5×10⁻⁴, P_SNP2 = 5×10⁻⁴, P_SNP3 = 4×10⁻³ and P_SNP4 = 0.025. The four SNPs were not correlated (0.01≤r²≤0.20). Haplotype analysis (excluding rare SNP2) revealed increasing PD risk with increasing risk alleles from OR = 1.27, P = 5×10⁻³ for one risk allele to OR = 1.65, P = 4×10⁻⁸ for three. Using additional 843 cases and 856 controls we replicated the independent effects of SNP1 (P_{conditioned-on-SNP4} = 0.04) and SNP4 (P_{conditioned-on-SNP1} = 0.04); SNP2 and SNP3 could not be replicated. In pooled GWAS and replication, SNP1 had OR_{conditioned-on-SNP4} = 1.23, P_{conditioned-on-SNP4} = 6×10⁻⁷; SNP4 had OR_{conditioned-on-SNP1} = 1.18, P_{conditioned-on-SNP1} = 3×10⁻³; and the haplotype with both risk alleles had OR = 1.48, P = 2×10⁻¹². Genotypic OR increased with the number of risk alleles an individual possessed up to OR = 1.94, P = 2×10⁻¹¹ for individuals who were homozygous for the risk allele at both SNP1 and SNP4. SNP1 is a variant in HLA-DRA and is associated with HLA-DRA, DRB5 and DQA2 gene expression. SNP4 is correlated (r² = 0.95) with variants that are associated with HLA-DQA2 expression, and with the top HLA SNP from the IPDGC GWAS (r² = 0.60). Our findings suggest more than one PD-HLA association; either different alleles of the same gene, or separate loci.     

Genetic determinants of serum testosterone concentrations in men

Testosterone concentrations in men are associated with cardiovascular morbidity, osteoporosis, and mortality and are affected by age, smoking, and obesity. Because of serum testosterone''s high heritability, we performed a meta-analysis of genome-wide association data in 8,938 men from seven cohorts and followed up the genome-wide significant findings in one in silico (n = 871) and two de novo replication cohorts (n = 4,620) to identify genetic loci significantly associated with serum testosterone concentration in men. All these loci were also associated with low serum testosterone concentration defined as <300 ng/dl. Two single-nucleotide polymorphisms at the sex hormone-binding globulin (SHBG) locus (17p13-p12) were identified as independently associated with serum testosterone concentration (rs12150660, p = 1.2×10⁻⁴¹ and rs6258, p = 2.3×10⁻²²). Subjects with ≥3 risk alleles of these variants had 6.5-fold higher risk of having low serum testosterone than subjects with no risk allele. The rs5934505 polymorphism near FAM9B on the X chromosome was also associated with testosterone concentrations (p = 5.6×10⁻¹⁶). The rs6258 polymorphism in exon 4 of SHBG affected SHBG''s affinity for binding testosterone and the measured free testosterone fraction (p<0.01). Genetic variants in the SHBG locus and on the X chromosome are associated with a substantial variation in testosterone concentrations and increased risk of low testosterone. rs6258 is the first reported SHBG polymorphism, which affects testosterone binding to SHBG and the free testosterone fraction and could therefore influence the calculation of free testosterone using law-of-mass-action equation.     

A SEL1L mutation links a canine progressive early-onset cerebellar ataxia to the endoplasmic reticulum-associated protein degradation (ERAD) machinery

Inherited ataxias are characterized by degeneration of the cerebellar structures, which results in progressive motor incoordination. Hereditary ataxias occur in many species, including humans and dogs. Several mutations have been found in humans, but the genetic background has remained elusive in dogs. The Finnish Hound suffers from an early-onset progressive cerebellar ataxia. We have performed clinical, pathological, and genetic studies to describe the disease phenotype and to identify its genetic cause. Neurological examinations on ten affected dogs revealed rapidly progressing generalized cerebellar ataxia, tremors, and failure to thrive. Clinical signs were present by the age of 3 months, and cerebellar shrinkage was detectable through MRI. Pathological and histological examinations indicated cerebellum-restricted neurodegeneration. Marked loss of Purkinje cells was detected in the cerebellar cortex with secondary changes in other cortical layers. A genome-wide association study in a cohort of 31 dogs mapped the ataxia gene to a 1.5 Mb locus on canine chromosome 8 (p_raw = 1.1×10⁻⁷, p_genome = 7.5×10⁻⁴). Sequencing of a functional candidate gene, sel-1 suppressor of lin-12-like (SEL1L), revealed a homozygous missense mutation, c.1972T>C; p.Ser658Pro, in a highly conserved protein domain. The mutation segregated fully in the recessive pedigree, and a 10% carrier frequency was indicated in a population cohort. SEL1L is a component of the endoplasmic reticulum (ER)–associated protein degradation (ERAD) machinery and has not been previously associated to inherited ataxias. Dysfunctional protein degradation is known to cause ER stress, and we found a significant increase in expression of nine ER stress responsive genes in the cerebellar cortex of affected dogs, supporting the pathogenicity of the mutation. Our study describes the first early-onset neurodegenerative ataxia mutation in dogs, establishes an ERAD–mediated neurodegenerative disease model, and proposes SEL1L as a new candidate gene in progressive childhood ataxias. Furthermore, our results have enabled the development of a genetic test for breeders.     

A comprehensive evaluation of potential lung function associated genes in the SpiroMeta general population sample

Rationale

Lung function measures are heritable traits that predict population morbidity and mortality and are essential for the diagnosis of chronic obstructive pulmonary disease (COPD). Variations in many genes have been reported to affect these traits, but attempts at replication have provided conflicting results. Recently, we undertook a meta-analysis of Genome Wide Association Study (GWAS) results for lung function measures in 20,288 individuals from the general population (the SpiroMeta consortium).

Objectives

To comprehensively analyse previously reported genetic associations with lung function measures, and to investigate whether single nucleotide polymorphisms (SNPs) in these genomic regions are associated with lung function in a large population sample.

Methods

We analysed association for SNPs tagging 130 genes and 48 intergenic regions (+/−10 kb), after conducting a systematic review of the literature in the PubMed database for genetic association studies reporting lung function associations.

Results

The analysis included 16,936 genotyped and imputed SNPs. No loci showed overall significant association for FEV₁ or FEV₁/FVC traits using a carefully defined significance threshold of 1.3×10⁻⁵. The most significant loci associated with FEV₁ include SNPs tagging MACROD2 (P = 6.81×10⁻⁵), CNTN5 (P = 4.37×10⁻⁴), and TRPV4 (P = 1.58×10⁻³). Among ever-smokers, SERPINA1 showed the most significant association with FEV₁ (P = 8.41×10⁻⁵), followed by PDE4D (P = 1.22×10⁻⁴). The strongest association with FEV₁/FVC ratio was observed with ABCC1 (P = 4.38×10⁻⁴), and ESR1 (P = 5.42×10⁻⁴) among ever-smokers.

Conclusions

Polymorphisms spanning previously associated lung function genes did not show strong evidence for association with lung function measures in the SpiroMeta consortium population. Common SERPINA1 polymorphisms may affect FEV₁ among smokers in the general population.     

Genome-wide association meta-analysis of cortical bone mineral density unravels allelic heterogeneity at the RANKL locus and potential pleiotropic effects on bone

Previous genome-wide association (GWA) studies have identified SNPs associated with areal bone mineral density (aBMD). However, this measure is influenced by several different skeletal parameters, such as periosteal expansion, cortical bone mineral density (BMD_C) cortical thickness, trabecular number, and trabecular thickness, which may be under distinct biological and genetic control. We have carried out a GWA and replication study of BMD_C, as measured by peripheral quantitative computed tomography (pQCT), a more homogenous and valid measure of actual volumetric bone density. After initial GWA meta-analysis of two cohorts (ALSPAC n = 999, aged ∼15 years and GOOD n = 935, aged ∼19 years), we attempted to replicate the BMD_C associations that had p<1×10⁻⁵ in an independent sample of ALSPAC children (n = 2803) and in a cohort of elderly men (MrOS Sweden, n = 1052). The rs1021188 SNP (near RANKL) was associated with BMD_C in all cohorts (overall p = 2×10⁻¹⁴, n = 5739). Each minor allele was associated with a decrease in BMD_C of ∼0.14SD. There was also evidence for an interaction between this variant and sex (p = 0.01), with a stronger effect in males than females (at age 15, males −6.77mg/cm³ per C allele, p = 2×10⁻⁶; females −2.79 mg/cm³ per C allele, p = 0.004). Furthermore, in a preliminary analysis, the rs1021188 minor C allele was associated with higher circulating levels of sRANKL (p<0.005). We show this variant to be independent from the previously aBMD associated SNP (rs9594738) and possibly from a third variant in the same RANKL region, which demonstrates important allelic heterogeneity at this locus. Associations with skeletal parameters reflecting bone dimensions were either not found or were much less pronounced. This finding implicates RANKL as a locus containing variation associated with volumetric bone density and provides further insight into the mechanism by which the RANK/RANKL/OPG pathway may be involved in skeletal development.     

The fate of assimilated carbon during drought: impacts on respiration in Amazon rainforests

Interannual variations in CO₂ exchange across Amazonia, as deduced from atmospheric inversions, correlate with El Niño occurrence. They are thought to result from changes in net ecosystem exchange and fire incidence that are both related to drought intensity. Alterations to net ecosystem production (NEP) are caused by changes in gross primary production (GPP) and ecosystem respiration (R_eco). Here, we analyse observations of the components of R_eco (leaves, live and dead woody tissue, and soil) to provide first estimates of changes in R_eco during short-term (seasonal to interannual) moisture limitation. Although photosynthesis declines if moisture availability is limiting, leaf dark respiration is generally maintained, potentially acclimating upwards in the longer term. If leaf area is lost, then short-term canopy-scale respiratory effluxes from wood and leaves are likely to decline. Using a moderate short-term drying scenario where soil moisture limitation leads to a loss of 0.5 m² m⁻² yr⁻¹ in leaf area index, we estimate a reduction in respiratory CO₂ efflux from leaves and live woody tissue of 1.0 (±0.4) t C ha⁻¹ yr⁻¹. Necromass decomposition declines during drought, but mortality increases; the median mortality increase following a strong El Niño is 1.1% (n=46 tropical rainforest plots) and yields an estimated net short-term increase in necromass CO₂ efflux of 0.13–0.18 t C ha⁻¹ yr⁻¹. Soil respiration is strongly sensitive to moisture limitation over the short term, but not to associated temperature increases. This effect is underestimated in many models but can lead to estimated reductions in CO₂ efflux of 2.0 (±0.5) t C ha⁻¹ yr⁻¹. Thus, the majority of short-term respiratory responses to drought point to a decline in R_eco, an outcome that contradicts recent regional-scale modelling of NEP. NEP varies with both GPP and R_eco but robust moisture response functions are clearly needed to improve quantification of the role of R_eco in influencing regional-scale CO₂ emissions from Amazonia.     

Genome-wide association study identifies HLA-DP as a susceptibility gene for pediatric asthma in Asian populations

Asthma is a complex phenotype influenced by genetic and environmental factors. We conducted a genome-wide association study (GWAS) with 938 Japanese pediatric asthma patients and 2,376 controls. Single-nucleotide polymorphisms (SNPs) showing strong associations (P<1×10⁻⁸) in GWAS were further genotyped in an independent Japanese samples (818 cases and 1,032 controls) and in Korean samples (835 cases and 421 controls). SNP rs987870, located between HLA-DPA1 and HLA-DPB1, was consistently associated with pediatric asthma in 3 independent populations (P _combined = 2.3×10⁻¹⁰, odds ratio [OR] = 1.40). HLA-DP allele analysis showed that DPA1*0201 and DPB1*0901, which were in strong linkage disequilibrium, were strongly associated with pediatric asthma (DPA1*0201: P = 5.5×10⁻¹⁰, OR = 1.52, and DPB1*0901: P = 2.0×10⁻⁷, OR = 1.49). Our findings show that genetic variants in the HLA-DP locus are associated with the risk of pediatric asthma in Asian populations.     

Novel loci for adiponectin levels and their influence on type 2 diabetes and metabolic traits: a multi-ethnic meta-analysis of 45,891 individuals

Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.5×10⁻⁸–1.2×10⁻⁴³). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after accounting for multiple testing (p<3×10⁻⁴). We next developed a multi-SNP genotypic risk score to test the association of adiponectin decreasing risk alleles on metabolic traits and diseases using consortia-level meta-analytic data. This risk score was associated with increased risk of T2D (p = 4.3×10⁻³, n = 22,044), increased triglycerides (p = 2.6×10⁻¹⁴, n = 93,440), increased waist-to-hip ratio (p = 1.8×10⁻⁵, n = 77,167), increased glucose two hours post oral glucose tolerance testing (p = 4.4×10⁻³, n = 15,234), increased fasting insulin (p = 0.015, n = 48,238), but with lower in HDL-cholesterol concentrations (p = 4.5×10⁻¹³, n = 96,748) and decreased BMI (p = 1.4×10⁻⁴, n = 121,335). These findings identify novel genetic determinants of adiponectin levels, which, taken together, influence risk of T2D and markers of insulin resistance.     

Genetic Modulation of Lipid Profiles following Lifestyle Modification or Metformin Treatment: The Diabetes Prevention Program

Weight-loss interventions generally improve lipid profiles and reduce cardiovascular disease risk, but effects are variable and may depend on genetic factors. We performed a genetic association analysis of data from 2,993 participants in the Diabetes Prevention Program to test the hypotheses that a genetic risk score (GRS) based on deleterious alleles at 32 lipid-associated single-nucleotide polymorphisms modifies the effects of lifestyle and/or metformin interventions on lipid levels and nuclear magnetic resonance (NMR) lipoprotein subfraction size and number. Twenty-three loci previously associated with fasting LDL-C, HDL-C, or triglycerides replicated (P = 0.04–1×10⁻¹⁷). Except for total HDL particles (r = −0.03, P = 0.26), all components of the lipid profile correlated with the GRS (partial |r| = 0.07–0.17, P = 5×10⁻⁵–1×10⁻¹⁹). The GRS was associated with higher baseline-adjusted 1-year LDL cholesterol levels (β = +0.87, SEE±0.22 mg/dl/allele, P = 8×10⁻⁵, P _interaction = 0.02) in the lifestyle intervention group, but not in the placebo (β = +0.20, SEE±0.22 mg/dl/allele, P = 0.35) or metformin (β = −0.03, SEE±0.22 mg/dl/allele, P = 0.90; P _interaction = 0.64) groups. Similarly, a higher GRS predicted a greater number of baseline-adjusted small LDL particles at 1 year in the lifestyle intervention arm (β = +0.30, SEE±0.012 ln nmol/L/allele, P = 0.01, P _interaction = 0.01) but not in the placebo (β = −0.002, SEE±0.008 ln nmol/L/allele, P = 0.74) or metformin (β = +0.013, SEE±0.008 nmol/L/allele, P = 0.12; P _interaction = 0.24) groups. Our findings suggest that a high genetic burden confers an adverse lipid profile and predicts attenuated response in LDL-C levels and small LDL particle number to dietary and physical activity interventions aimed at weight loss.     

Common variants in CRP and LEPR influence high sensitivity C-reactive protein levels in North Indians

Background

High sensitivity C-reactive protein (hsCRP) levels are shown to be influenced by genetic variants in Europeans; however, little is explored in Indian population.

Methods

Herein, we comprehensively evaluated association of all previously reported genetic determinants of hsCRP levels, including 18 cis (proximal to CRP gene) and 73 trans-acting (distal to CRP gene) variants in 4,200 North Indians of Indo-European ethnicity. First, we evaluated association of 91 variants from 12 candidate loci with hsCRP levels in 2,115 North Indians (1,042 non-diabetic subjects and 1,073 patients with type 2 diabetes). Then, cis and trans-acting variants contributing maximally to hsCRP level variation were further replicated in an independent 2,085 North Indians (1,047 patients with type 2 diabetes and 1,038 non-diabetic subjects).

Results

We found association of 12 variants from CRP, LEPR, IL1A, IL6, and IL6R with hsCRP levels in non-diabetic subjects. However, only rs3093059-CRP [β = 0.33, P = 9.6×10⁻⁵] and the haplotype harboring rs3093059 risk allele [β = 0.32 µg/mL, P = 1.4×10⁻⁴/P_perm = 9.0×10⁻⁴] retained significance after correcting for multiple testing. The cis-acting variant rs3093059-CRP had maximum contribution to the variance in hsCRP levels (1.14%). Among, trans-acting variants, rs1892534-LEPR was observed to contribute maximally to hsCRP level variance (0.59%). Associations of rs3093059-CRP and rs1892534-LEPR were confirmed by replication and attained higher significance after meta-analysis [β_meta = 0.26/0.22; P_meta = 4.3×10⁻⁷/7.4×10⁻³ and β_meta = −0.15/−0.12; P_meta = 2.0×10⁻⁶/1.6×10⁻⁶ for rs3093059 and rs1892534, respectively in non-diabetic subjects and all subjects taken together].

Conclusion

In conclusion, we identified rs3093059 in CRP and rs1892534 in LEPR as major cis and trans-acting contributor respectively, to the variance in hsCRP levels in North Indian population.     

Smoking and COX-2 functional polymorphisms interact to increase the risk of gastric cardia adenocarcinoma in Chinese population

Background

Over-expression and increased activity of cyclooxygenase (COX)-2 induced by smoking has been implicated in the development of cancer. This study aimed to explore the interaction between smoking and functional polymorphisms of COX-2 in modulation of gastric cardia adenocarcinoma (GCA) risk.

Methods and Findings

Three COX-2 polymorphisms, including –1195G>A (rs689466), –765G>C (rs20417), and 587Gly>Arg (rs3218625), were genotyped in 357 GCA patients and 985 controls. In the multivariate logistic regression analysis, we found that the –1195AA, –765GC, and 587Arg/Arg genotypes were associated with increased risk of GCA (OR = 1.50, 95% CI = 1.05–2.13; OR = 2.06, 95% CI = 1.29–3.29 and OR = 1.67, 95% CI = 1.04–2.66, respectively). Haplotype association analysis showed that compared with G₋₁₁₉₅-G₋₇₆₅- G_Gly587Arg, the A₋₁₁₉₅-C₋₇₆₅-A_Gly587Arg conferred an increased risk of GCA (OR = 2.49, 95% CI = 1.54–4.01). Moreover, significant multiplicative interactions were observed between smoking and these three polymorphisms of –1195G>A, –765G>C, and 587Gly>Arg, even after correction by false discovery rate (FDR) method for multiple comparisons (FDR-P _interaction = 0.006, 5.239×10⁻⁴ and 0.017, respectively). Similarly, haplotypes incorporating these three polymorphisms also showed significant interaction with smoking in the development of GCA (P for multiplicative interaction = 2.65×10⁻⁶).

Conclusion

These findings indicated that the functional polymorphisms of COX-2, in interaction with smoking, may play a substantial role in the development of GCA.     

A new estimation of global soil greenhouse gas fluxes using a simple data-oriented model

Soil greenhouse gas fluxes (particularly CO₂, CH₄, and N₂O) play important roles in climate change. However, despite the importance of these soil greenhouse gases, the number of reports on global soil greenhouse gas fluxes is limited. Here, new estimates are presented for global soil CO₂ emission (total soil respiration), CH₄ uptake, and N₂O emission fluxes, using a simple data-oriented model. The estimated global fluxes for CO₂ emission, CH₄ uptake, and N₂O emission were 78 Pg C yr⁻¹ (Monte Carlo 95% confidence interval, 64–95 Pg C yr⁻¹), 18 Tg C yr⁻¹ (11–23 Tg C yr⁻¹), and 4.4 Tg N yr⁻¹ (1.4–11.1 Tg N yr⁻¹), respectively. Tropical regions were the largest contributor of all of the gases, particularly the CO₂ and N₂O fluxes. The soil CO₂ and N₂O fluxes had more pronounced seasonal patterns than the soil CH₄ flux. The collected estimates, including both the previous and the present estimates, demonstrate that the means of the best estimates from each study were 79 Pg C yr⁻¹ (291 Pg CO₂ yr⁻¹; coefficient of variation, CV = 13%, N = 6) for CO₂, 21 Tg C yr⁻¹ (29 Tg CH₄ yr⁻¹; CV = 24%, N = 24) for CH₄, and 7.8 Tg N yr⁻¹ (12.2 Tg N₂O yr⁻¹; CV = 38%, N = 11) for N₂O. For N₂O, the mean of the estimates that was calculated by excluding the earliest two estimates was 6.6 Tg N yr⁻¹ (10.4 Tg N₂O yr⁻¹; CV = 22%, N = 9). The reported estimates vary and have large degrees of uncertainty but their overall magnitudes are in general agreement. To further minimize the uncertainty of soil greenhouse gas flux estimates, it is necessary to build global databases and identify key processes in describing global soil greenhouse gas fluxes.     

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.     

Comparison of 6q25 Breast Cancer Hits from Asian and European Genome Wide Association Studies in the Breast Cancer Association Consortium (BCAC)

The 6q25.1 locus was first identified via a genome-wide association study (GWAS) in Chinese women and marked by single nucleotide polymorphism (SNP) rs2046210, approximately 180 Kb upstream of ESR1. There have been conflicting reports about the association of this locus with breast cancer in Europeans, and a GWAS in Europeans identified a different SNP, tagged here by rs12662670. We examined the associations of both SNPs in up to 61,689 cases and 58,822 controls from forty-four studies collaborating in the Breast Cancer Association Consortium, of which four studies were of Asian and 39 of European descent. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI). Case-only analyses were used to compare SNP effects in Estrogen Receptor positive (ER+) versus negative (ER−) tumours. Models including both SNPs were fitted to investigate whether the SNP effects were independent. Both SNPs are significantly associated with breast cancer risk in both ethnic groups. Per-allele ORs are higher in Asian than in European studies [rs2046210: OR (A/G) = 1.36 (95% CI 1.26–1.48), p = 7.6×10⁻¹⁴ in Asians and 1.09 (95% CI 1.07–1.11), p = 6.8×10⁻¹⁸ in Europeans. rs12662670: OR (G/T) = 1.29 (95% CI 1.19–1.41), p = 1.2×10⁻⁹ in Asians and 1.12 (95% CI 1.08–1.17), p = 3.8×10⁻⁹ in Europeans]. SNP rs2046210 is associated with a significantly greater risk of ER− than ER+ tumours in Europeans [OR (ER−) = 1.20 (95% CI 1.15–1.25), p = 1.8×10⁻¹⁷ versus OR (ER+) = 1.07 (95% CI 1.04–1.1), p = 1.3×10⁻⁷, p_{heterogeneity} = 5.1×10⁻⁶]. In these Asian studies, by contrast, there is no clear evidence of a differential association by tumour receptor status. Each SNP is associated with risk after adjustment for the other SNP. These results suggest the presence of two variants at 6q25.1 each independently associated with breast cancer risk in Asians and in Europeans. Of these two, the one tagged by rs2046210 is associated with a greater risk of ER− tumours.     

The PNPLA3 rs738409 G-allele associates with reduced fasting serum triglyceride and serum cholesterol in Danes with impaired glucose regulation

Background and Aim

Non-alcoholic fatty liver disease (NAFLD) is a common condition, associated with hepatic insulin resistance and the metabolic syndrome including hyperglycaemia and dyslipidemia. We aimed at studying the potential impact of the NAFLD-associated PNPLA3 rs738409 G-allele on NAFLD-related metabolic traits in hyperglycaemic individuals.

Methods

The rs738409 variant was genotyped in the population-based Inter99 cohort examined by an oral glucose-tolerance test, and a combined study-sample consisting of 192 twins (96 twin pairs) and a sub-set of the Inter99 population (n = 63) examined by a hyperinsulinemic euglycemic clamp (n _total = 255). In Inter99, we analyzed associations of rs738409 with components of the WHO-defined metabolic syndrome (n = 5,847) and traits related to metabolic disease (n = 5,663). In the combined study sample we elucidated whether the rs738409 G-allele altered hepatic or peripheral insulin sensitivity. Study populations were divided into individuals with normal glucose-tolerance (NGT) and with impaired glucose regulation (IGR).

Results

The case-control study showed no associations with components of the metabolic syndrome or the metabolic syndrome. Among 1,357 IGR individuals, the rs738409 G-allele associated with decreased fasting serum triglyceride levels (per allele effect(β) = −9.9% [−14.4%;−4.0% (95% CI)], p = 5.1×10⁻⁵) and fasting total cholesterol (β = −0.2 mmol/l [−0.3;−0.01 mmol/l(95% CI)], p = 1.5×10⁻⁴). Meta-analyses showed no impact on hepatic or peripheral insulin resistance in carriers of the rs738409 G-allele.

Conclusion

Our findings suggest that the G-allele of PNPLA3 rs738409 associates with reduced fasting levels of cholesterol and triglyceride in individuals with IGR.