Kristoffer Sølvsten Burgdorf

A human gut microbial gene catalogue established by metagenomic sequencing

To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, ∼150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.

Richness of human gut microbiome correlates with metabolic markers

We are facing a global metabolic health crisis provoked by an obesity epidemic. Here we report the human gut microbial composition in a population sample of 123 non-obese and 169 obese Danish individuals. We find two groups of individuals that differ by the number of gut microbial genes and thus gut bacterial richness. They contain known and previously unknown bacterial species at different proportions; individuals with a low bacterial richness (23% of the population) are characterized by more marked overall adiposity, insulin resistance and dyslipidaemia and a more pronounced inflammatory phenotype when compared with high bacterial richness individuals. The obese individuals among the lower bacterial richness group also gain more weight over time. Only a few bacterial species are sufficient to distinguish between individuals with high and low bacterial richness, and even between lean and obese participants. Our classifications based on variation in the gut microbiome identify subsets of individuals in the general white adult population who may be at increased risk of progressing to adiposity-associated co-morbidities.

Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes.

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

Roux-en-Y gastric bypass surgery of morbidly obese patients induces swift and persistent changes of the individual gut microbiota

BackgroundRoux-en-Y gastric bypass (RYGB) is an effective means to achieve sustained weight loss for morbidly obese individuals. Besides rapid weight reduction, patients achieve major improvements of insulin sensitivity and glucose homeostasis. Dysbiosis of gut microbiota has been associated with obesity and some of its co-morbidities, like type 2 diabetes, and major changes of gut microbial communities have been hypothesized to mediate part of the beneficial metabolic effects observed after RYGB. Here we describe changes in gut microbial taxonomic composition and functional potential following RYGB.MethodsWe recruited 13 morbidly obese patients who underwent RYGB, carefully phenotyped them, and had their gut microbiomes quantified before (nu2009=u200913) and 3xa0months (nu2009=u200912) and 12xa0months (nu2009=u20098) after RYGB. Following shotgun metagenomic sequencing of the fecal microbial DNA purified from stools, we characterized the gut microbial composition at species and gene levels followed by functional annotation.ResultsIn parallel with the weight loss and metabolic improvements, gut microbial diversity increased within the first 3xa0months after RYGB and remained high 1xa0year later. RYGB led to altered relative abundances of 31 species (Pu2009<u20090.05, qu2009<u20090.15) within the first 3xa0months, including those of Escherichia coli, Klebsiella pneumoniae, Veillonella spp., Streptococcus spp., Alistipes spp., and Akkermansia muciniphila. Sixteen of these species maintained their altered relative abundances during the following 9xa0months. Interestingly, Faecalibacterium prausnitzii was the only species that decreased in relative abundance. Fifty-three microbial functional modules increased their relative abundance between baseline and 3xa0months (Pu2009<u20090.05, qu2009<u20090.17). These functional changes included increased potential (i) to assimilate multiple energy sources using transporters and phosphotransferase systems, (ii) to use aerobic respiration, (iii) to shift from protein degradation to putrefaction, and (iv) to use amino acids and fatty acids as energy sources.ConclusionsWithin 3xa0months after morbidly obese individuals had undergone RYGB, their gut microbiota featured an increased diversity, an altered composition, an increased potential for oxygen tolerance, and an increased potential for microbial utilization of macro- and micro-nutrients. These changes were maintained for the first year post-RYGB.Trial registrationCurrent controlled trials (ID NCT00810823, NCT01579981, and NCT01993511).

AHSG Tag Single Nucleotide Polymorphisms Associate With Type 2 Diabetes and Dyslipidemia: Studies of Metabolic Traits in 7,683 White Danish Subjects

OBJECTIVE—The gene encoding the α2 Heremans-Schmid glycoprotein (AHSG) is a credible biological and positional candidate gene for type 2 diabetes and the metabolic syndrome, and previous attempts to relate AHSG variation with type 2 diabetes and obesity in Swedish and French Caucasians have been largely successful. We related seven frequent AHSG tag single nucleotide polymorphisms to a range of metabolic traits, including type 2 diabetes, obesity, and dyslipidemia. RESEARCH DESIGN AND METHODS—The polymorphisms were genotyped in 7,683 white Danish subjects using Taqman allelic discrimination or chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, providing a statistical power of >99% to replicate previous findings. Data were analyzed in case-control and haplotype settings, and quantitative metabolic traits were examined for association. Moreover, epistatic effects between AHSG variants and insulin receptor substrate-1 (IRS1) and β-2-adrenergic receptor polymorphisms were investigated. RESULTS—The −469T>G (rs2077119) and IVS6+98C>T (rs2518136) polymorphisms were associated with type 2 diabetes (P = 0.007 and P = 0.006, respectively, or Pcorr = 0.04 and Pcorr = 0.03, respectively, following correction for multiple hypothesis testing), and in a combined analysis of the present and a previous study −469T>G remained significant (odds ratio 0.90 [95% CI 0.84–0.97]; P = 0.007). Furthermore, two AHSG haplotypes were associated with dyslipidemia (P = 0.003 and Pcorr = 0.009). Thr248Met (rs4917) tended to associate with lower fasting and post–oral glucose tolerance test serum insulin release (P = 0.02, Pcorr = 0.1 for fasting and P = 0.04, Pcorr = 0.2 for area under the insulin curve) and improved insulin sensitivity estimated by the homeostasis model assessment of insulin resistance (9.0 vs. 8.6 mmol · l−1 · pmol−1 · l−1; P = 0.01, Pcorr = 0.06). Indications of epistatic effects of AHSG variants with the IRS1 Gly971Arg polymorphism were observed for fasting serum triglyceride concentrations. CONCLUSIONS—Based on present and previous findings, common variation in AHSG may contribute to the interindividual variation in metabolic traits.

Variation in the gene encoding Krüppel-like factor 7 influences body fat: studies of 14 818 Danes

OBJECTIVEnKLF7 encodes Krüppel-like factor (KLF) 7, a member of the KLF family of transcription factors, initially shown to play important roles in cellular development and differentiation, and reported to be specifically involved in adipogenesis. Several single nucleotide polymorphisms (SNPs) have been identified in KLF7, of which the A-allele of rs2302870 has been associated with type 2 diabetes in a Japanese population; however, a possible association of KLF7 SNPs with obesity has not been investigated. We aimed to identify variation in the putative promoter region, the coding regions, exon/intron boundaries, and 3-UTR of KLF7, and to examine identified variants in relation to obesity, type 2 diabetes, and related quantitative traits in Danish individuals.nnnMETHODSnIdentified variants were investigated for association with type 2 diabetes in 8777 individuals and with obesity in 14 818 individuals.nnnRESULTSnWe identified four common SNPs in low pairwise linkage disequilibrium; three in the putative promoter region (-1119 G>A, -963 C>A (rs7568369), and -614 G>A) and IVS2+35092 A>C (rs2302870). We failed to confirm an association between rs2302870 and type 2 diabetes. Neither was rs7568369 associated with type 2 diabetes; however, the minor A-allele of rs7568369 protected against obesity (OR=0.90 (0.84-0.96), P=0.001) and in studies of quantitative traits (n=5,535) the variant associated with decreased body mass index (P=0.002) and waist circumference (P=0.003). The -1119 G>A and -614 G>A variants were not associated with obesity or type 2 diabetes.nnnCONCLUSIONnWe identified a novel association between the minor A-allele of KLF7 rs7568369 and protection against obesity in the Danish population.

AHSG tagSNPs associate with type 2 diabetes and dyslipidemia: studies of metabolic traits in 7,683 Danish whites

OBJECTIVE—The gene encoding the α2 Heremans-Schmid glycoprotein (AHSG) is a credible biological and positional candidate gene for type 2 diabetes and the metabolic syndrome, and previous attempts to relate AHSG variation with type 2 diabetes and obesity in Swedish and French Caucasians have been largely successful. We related seven frequent AHSG tag single nucleotide polymorphisms to a range of metabolic traits, including type 2 diabetes, obesity, and dyslipidemia. RESEARCH DESIGN AND METHODS—The polymorphisms were genotyped in 7,683 white Danish subjects using Taqman allelic discrimination or chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, providing a statistical power of >99% to replicate previous findings. Data were analyzed in case-control and haplotype settings, and quantitative metabolic traits were examined for association. Moreover, epistatic effects between AHSG variants and insulin receptor substrate-1 (IRS1) and β-2-adrenergic receptor polymorphisms were investigated. RESULTS—The −469T>G (rs2077119) and IVS6+98C>T (rs2518136) polymorphisms were associated with type 2 diabetes (P = 0.007 and P = 0.006, respectively, or Pcorr = 0.04 and Pcorr = 0.03, respectively, following correction for multiple hypothesis testing), and in a combined analysis of the present and a previous study −469T>G remained significant (odds ratio 0.90 [95% CI 0.84–0.97]; P = 0.007). Furthermore, two AHSG haplotypes were associated with dyslipidemia (P = 0.003 and Pcorr = 0.009). Thr248Met (rs4917) tended to associate with lower fasting and post–oral glucose tolerance test serum insulin release (P = 0.02, Pcorr = 0.1 for fasting and P = 0.04, Pcorr = 0.2 for area under the insulin curve) and improved insulin sensitivity estimated by the homeostasis model assessment of insulin resistance (9.0 vs. 8.6 mmol · l−1 · pmol−1 · l−1; P = 0.01, Pcorr = 0.06). Indications of epistatic effects of AHSG variants with the IRS1 Gly971Arg polymorphism were observed for fasting serum triglyceride concentrations. CONCLUSIONS—Based on present and previous findings, common variation in AHSG may contribute to the interindividual variation in metabolic traits.

Association studies of novel obesity-related gene variants with quantitative metabolic phenotypes in a population-based sample of 6,039 Danish individuals

Aims/hypothesisGenome-wide association studies have identified novel WHR and BMI susceptibility loci. The aim of this study was to elucidate if any of these loci had an effect on quantitative measures of glucose homeostasis, including estimates of insulin release and insulin sensitivity in an epidemiological setting.MethodsBy applying an additive genetic model, 14 WHR-associated gene variants and 18 BMI-associated variants were investigated for their relationships with glucose-related metabolic traits in treatment-naive individuals from the population-based Inter99 study sample (nu2009=u20096,039).ResultsOf the variants associated with BMI, the QPCTL rs2287019 C allele was associated with an increased insulinogenic index of 7.4% per risk allele (pu2009=u20094.0u2009×u200910−7) and increased disposition index of 5.6% (pu2009=u20096.4u2009×u200910−5). The LRP1B rs2890652 C allele was associated with insulin resistance, showing a 3.3% increase (pu2009=u20090.0011) using the HOMA-insulin resistance (HOMA-IR) index and a 2.2% reduction (pu2009=u20090.0014) with the Matsuda index. Of the variants associated with WHR, LYPLAL1/SLC30A10 rs4846567 G allele carriers showed a 5.2% lower HOMA-IR (pu2009=u20090.00086) in women, indicating improved insulin sensitivity. Female carriers of the VEGFA rs6905288 A allele were insulin resistant, with a 3.7% increase in HOMA-IR (pu2009=u20090.00036) and 4.0% decrease in Matsuda index (pu2009=u20092u2009×u200910−4).ConclusionsOur correlative findings from analysing single-locus data suggest that some variation in validated BMI and WHR loci are associated with either increased or decreased insulin sensitivity and thereby potentially with metabolically healthy or metabolically unhealthy subsets of obesity. The results call for testing in larger study samples and for further physiological exploration of the possible metabolic implications of these loci.

The kcnmb1 Glu65lys polymorphism associates with reduced systolic and diastolic blood pressure in the Inter99 study of 5729 Danes

Objective The large Ca2+ and voltage-dependent potassium channel is important in regulating vascular tone in smooth muscle tissue. The rs11739136 KCNMB1 Glu65Lys polymorphism in the β1 subunit of the Ca2+ and voltage-dependent potassium channel has, in some studies, been reported to associate with a protective effect on diastolic hypertension. The previous studies have, however, been conflicting, and the aim of the present study was to clarify the impact of the Glu65Lys polymorphism on hypertension at the population level of middle-aged people. Design Large-scale sex-stratified case–control studies and analyses of quantitative blood pressure. Methods The KCNMB1 Glu65Lys (rs11739136) polymorphism was genotyped in 5729 Danes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of PCR-generated primer extension products. Results In the population-based Inter99 cohort, the Glu65Lys polymorphism was associated with a 1.3% decrease in systolic blood pressure (P = 0.01) and a 1.1% decrease in diastolic blood pressure (P = 0.04) per Lys-allele among 2668 men. Among women, we observed no association between systolic or diastolic blood pressure and the Glu65Lys polymorphism. Conclusion If replicated, our findings suggest that the KCNMB1 Glu65Lys polymorphism associates with reduced systolic and diastolic blood pressure in middle-aged men.

Studies of association between LPIN1 variants and common metabolic phenotypes among 17 538 Danes.

OBJECTIVEnLipin-1, encoded by LPIN1, is expressed in the major metabolically active tissues. Decreased expression of lipin-1 in adipose tissue correlates with increased insulin resistance, and tagging of the LPIN1 locus has shown that rs33997857, rs6744682, and rs6708316 associate with metabolic phenotypes, specifically body mass index (BMI) and fasting serum lipid levels, both on the individual single-nucleotide polymorphism level and with a three-marker haplotype. Our aim was to validate the reported findings in the Danish population.nnnDESIGNnIn the present study, variants were analyzed in LPIN1 using case-control studies, haplotype analyses, and quantitative trait analyses in a population of 17,538 Danes.nnnMETHODSnThe three LPIN1 variants were genotyped in 17,538 Danes from four study populations of middle-aged people. This provided us with a statistical power >99% to replicate previous findings. Variants were analyzed individually and in haplotype combinations in studies of quantitative metabolic traits and in case-control studies.nnnRESULTSnNone of the three variants were associated with the examined quantitative traits including BMI, waist circumference, blood pressure, fasting serum lipid concentrations, or plasma glucose or serum insulin concentrations in the fasting state and following an oral glucose tolerance test. Haplotypes were tested for association with quantitative traits; however, only nominal association with blood pressure (P=0.04) and waist circumference (P=0.04) was observed. In case-control studies, no association was found for individual variants or the three-marker haplotype.nnnCONCLUSIONnLPIN1 rs33997857, rs6744682, and rs6708316 did not associate with type 2 diabetes, obesity, or related quantitative metabolic phenotypes in the Danish population examined.