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1.  Mutations in HNF1A Result in Marked Alterations of Plasma Glycan Profile 
Diabetes  2013;62(4):1329-1337.
A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction.
PMCID: PMC3609552  PMID: 23274891
2.  Chronic Family Stress Moderates the Association between a TOMM40 Variant and Triglyceride Levels in Two Independent Caucasian Samples 
Biological psychology  2013;93(1):184-189.
TOMM40 SNP rs157580 has been associated with triglyceride levels in Genome-wide association studies (GWAS). Chronic caregiving stress moderates the association between triglyceride levels and a nearby SNP rs439401 that is associated with triglyceride levels in GWAS. Here, we report data from two independent Caucasian samples (242 U.S. women and men; 466 Danish men) testing the hypothesis that chronic family stress also moderates the association between rs157580 and triglyceride levels. The interaction of rs157580 and family stress in predicting triglyceride levels was statistically significant in the U.S. sample (p = 0.004) and marginally significant (p = 0.075) in the Danish sample. The G allele of rs157580 was associated with increased triglyceride levels among family stressed cases in both samples compared with A/A cases, but not among controls. Chronic family stress moderates the association of rs157580 variants with triglyceride levels and should be taken into account for disease risk assessment and potential intervention.
PMCID: PMC3739426  PMID: 23435269
3.  Evaluation of a target region capture sequencing platform using monogenic diabetes as a study-model 
BMC Genetics  2014;15:13.
Monogenic diabetes is a genetic disease often caused by mutations in genes involved in beta-cell function. Correct sub-categorization of the disease is a prerequisite for appropriate treatment and genetic counseling. Target-region capture sequencing is a combination of genomic region enrichment and next generation sequencing which might be used as an efficient way to diagnose various genetic disorders. We aimed to develop a target-region capture sequencing platform to screen 117 selected candidate genes involved in metabolism for mutations and to evaluate its performance using monogenic diabetes as a study-model.
The performance of the assay was evaluated in 70 patients carrying known disease causing mutations previously identified in HNF4A, GCK, HNF1A, HNF1B, INS, or KCNJ11. Target regions with a less than 20-fold sequencing depth were either introns or UTRs. When only considering translated regions, the coverage was 100% with a 50-fold minimum depth. Among the 70 analyzed samples, 63 small size single nucleotide polymorphisms and indels as well as 7 large deletions and duplications were identified as being the pathogenic variants. The mutations identified by the present technique were identical with those previously identified through Sanger sequencing and Multiplex Ligation-dependent Probe Amplification.
We hereby demonstrated that the established platform as an accurate and high-throughput gene testing method which might be useful in the clinical diagnosis of monogenic diabetes.
PMCID: PMC3943834  PMID: 24476040
4.  Thromboprophylaxis only during hospitalisation in fast-track hip and knee arthroplasty, a prospective cohort study 
BMJ Open  2013;3(12):e003965.
International guidelines recommend thrombosis prophylaxis after total hip arthroplasty (THA) and total knee arthroplasty (TKA) for up to 35 days. However, previous studies often have hospital stays (length of stay; LOS) of 8–12 days and not considering early mobilisation, which may reduce incidence of venous thromboembolic events (VTE). We investigated the incidence of any symptomatic thromboembolic events (TEEs) with only in-hospital prophylaxis if LOS ≤5 days after fast-track THA and TKA.
A prospective descriptive multicentre cohort study in fast-track THA and TKA from February 2010 to December 2011, with complete 90-day follow-up through the Danish National Patient Registry and patient files.
6 Danish high-volume centres with a similar standardised fast-track setup, including spinal anaesthesia, opioid-sparing analgesia, early mobilisation, functional discharge criteria and discharge to own home.
4924 consecutive unselected unilateral primary THA and TKAs in patients ≥18 years with no preoperative use of continuous ‘potent’ anticoagulative therapy (vitamin K antagonists).
Prophylaxis with low-molecular-weight heparin or factor Xa-inhibitors only during hospitalisation when LOS ≤5 days.
Incidence of symptomatic TEE-related, VTE-related and VTE-related mortality ≤90 days postoperatively.
LOS ≤5 days and thromboprophylaxis only during hospitalisation occurred in 4659 procedures (94.6% of total). Median LOS and prophylaxis duration was 2 days (IQR 2–3) with 0.84% (95% CI 0.62% to 1.15%) TEE and 0.41% (0.26% to 0.64%) VTE during 90-day follow-up. VTE consisted of five pulmonary embolisms (0.11% (0.05% to 0.25%)) and 14 deep venous thrombosis (0.30% (0.18% to 0.50%)). There were four (0.09% (0.04% to 0.23%)) surgery-related deaths, of which 1 (0.02% (0.00% to 0.12%)) was due to pulmonary embolism, and 6 (0.13% (0.06% to 0.28%)) deaths of unknown causes after discharge.
The low incidence of TEE and VTE suggests that in-hospital prophylaxis only, is safe in fast-track THA and TKA patients with LOS of ≤5 days. Guidelines on thromboprophylaxis may need reconsideration in fast-track elective surgery.
Trial Registration NCT01557725
PMCID: PMC3863129  PMID: 24334158
5.  Joint Analysis of Individual Participants’ Data from 17 Studies on the Association of the IL6 Variant -174G>C with Circulating Glucose Levels, Interleukin-6 Levels, and Body-Mass Index 
Annals of medicine  2009;41(2):128-138.
Several studies have investigated associations between the -174G>C polymorphism (rs1800795) of the IL6-gene, but presented inconsistent results.
This joint analysis aimed to clarify whether IL6 -174G>C was associated with type 2 diabetes mellitus (T2DM) related quantitative phenotypes.
Individual-level data from all studies of the IL6-T2DM consortium on Caucasian subjects with available BMI were collected. As study-specific estimates did not show heterogeneity (P>0.1), they were combined by using the inverse-variance fixed-effect model.
The main analysis included 9440, 7398, 24,117, or 5659 nondiabetic and manifest T2DM subjects for fasting glucose, 2-hour glucose, BMI or circulating interleukin-6 levels, respectively. IL6 -174 C-allele carriers had significantly lower fasting glucose (−0.091mmol/L, P=0.014). There was no evidence for association between IL6 -174G>C and BMI or interleukin-6. In an additional analysis of 641 subjects known to develop T2DM later on, the IL6 -174 CC-genotype was associated with higher baseline interleukin-6 (+0.75pg/mL, P=0.004), which was consistent with higher interleukin-6 in the 966 manifest T2DM subjects (+0.50pg/mL, P=0.044).
Our data suggest association between IL6 -174G>C and quantitative glucose, and exploratory analysis indicated modulated interleukin-6 levels in pre-diabetic subjects, being in-line with this SNP’s previously reported T2DM association and a role of circulating interleukin-6 as intermediate phenotype.
PMCID: PMC3801210  PMID: 18752089
blood glucose; body mass index; diabetes mellitus; type 2; epidemiology; molecular; genes; inflammation mediators; interleukin-6; intermediate phenotype; meta-analysis; polymorphism; single nucleotide
6.  A human gut microbial gene catalog established by metagenomic sequencing 
Nature  2010;464(7285):59-65.
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 nonredundant microbial genes, derived from 576.7 Gb 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 microbial genes of the cohort and likely 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, suggesting that the entire cohort harbours between 1000 and 1150 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 encoded by the gene set.
PMCID: PMC3779803  PMID: 20203603
7.  Chronic Mild Hyperglycemia in GCK-MODY Patients Does Not Increase Carotid Intima-Media Thickness 
Aim. GCK-MODY is an autosomal dominant form of diabetes caused by heterozygous mutations in the glucokinase gene leading to a lifelong mild hyperglycemia. The risk of macrovascular complications is considered low, but studies are limited. We, therefore, investigated the carotid intima-media thickness (CIMT) as an indicator of macrovascular complications in a group of patients with GCK-MODY. Methods. Twenty-seven GCK mutation carriers and 24 controls recruited among their first-degree relatives were compared, all aging over 35 years. The CIMT was tested using a high-resolution B-mode carotid ultrasonography. Medical history, anthropometry, and biochemical blood workup were obtained. Results. The mean CIMT was 0.707 ± 0.215 mm (mean ± SD) in GCK mutation carriers and 0.690 ± 0.180 mm in control individuals. When adjusted for age, gender, and family status, the estimated mean difference in CIMT between the two groups increased to 0.049 mm (P = 0.19). No difference was detected for other characteristics, with the exception of fasting blood glucose (GCK-MODY 7.6 mmol/L ± 1.2 (136.4 mg/dL); controls 5.3 mmol/L ± 0.3 (95.4 mg/dL); P < 0.0001) and glycated hemoglobin HbA1c (GCK-MODY 6.9% ± 1.0%, 52 mmol/mol ± 10; controls 5.7% ± 0.4%, 39 mmol/mol ± 3; P < 0.0001). The frequency of myocardial infarction and ischemic stroke did not differ between groups. Conclusion. Our data indicate that the persistent hyperglycemia in GCK-MODY is associated with a low risk of developing diabetic macrovascular complications.
PMCID: PMC3786513  PMID: 24101925
8.  Enterotypes of the human gut microbiome 
Nature  2011;473(7346):174-180.
Our knowledge on species and function composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about their variation across the world. Combining 22 newly sequenced fecal metagenomes of individuals from 4 countries with previously published datasets, we identified three robust clusters (enterotypes hereafter) that are not nation or continent-specific. We confirmed the enterotypes also in two published, larger cohorts suggesting that intestinal microbiota variation is generally stratified, not continuous. This further indicates the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis for a community understanding. While individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.
PMCID: PMC3728647  PMID: 21508958
9.  Gene × Physical Activity Interactions in Obesity: Combined Analysis of 111,421 Individuals of European Ancestry 
PLoS Genetics  2013;9(7):e1003607.
Numerous obesity loci have been identified using genome-wide association studies. A UK study indicated that physical activity may attenuate the cumulative effect of 12 of these loci, but replication studies are lacking. Therefore, we tested whether the aggregate effect of these loci is diminished in adults of European ancestry reporting high levels of physical activity. Twelve obesity-susceptibility loci were genotyped or imputed in 111,421 participants. A genetic risk score (GRS) was calculated by summing the BMI-associated alleles of each genetic variant. Physical activity was assessed using self-administered questionnaires. Multiplicative interactions between the GRS and physical activity on BMI were tested in linear and logistic regression models in each cohort, with adjustment for age, age2, sex, study center (for multicenter studies), and the marginal terms for physical activity and the GRS. These results were combined using meta-analysis weighted by cohort sample size. The meta-analysis yielded a statistically significant GRS × physical activity interaction effect estimate (Pinteraction = 0.015). However, a statistically significant interaction effect was only apparent in North American cohorts (n = 39,810, Pinteraction = 0.014 vs. n = 71,611, Pinteraction = 0.275 for Europeans). In secondary analyses, both the FTO rs1121980 (Pinteraction = 0.003) and the SEC16B rs10913469 (Pinteraction = 0.025) variants showed evidence of SNP × physical activity interactions. This meta-analysis of 111,421 individuals provides further support for an interaction between physical activity and a GRS in obesity disposition, although these findings hinge on the inclusion of cohorts from North America, indicating that these results are either population-specific or non-causal.
Author Summary
We undertook analyses in 111,421 adults of European descent to examine whether physical activity diminishes the genetic risk of obesity predisposed by 12 single nucleotide polymorphisms, as previously reported in a study of 20,000 UK adults (Li et al, PLoS Med. 2010). Although the study by Li et al is widely cited, the original report has not been replicated to our knowledge. Therefore, we sought to confirm or refute the original study's findings in a combined analysis of 111,421 adults. Our analyses yielded a statistically significant interaction effect (Pinteraction = 0.015), confirming the original study's results; we also identified an interaction between the FTO locus and physical activity (Pinteraction = 0.003), verifying previous analyses (Kilpelainen et al, PLoS Med., 2010), and we detected a novel interaction between the SEC16B locus and physical activity (Pinteraction = 0.025). We also examined the power constraints of interaction analyses, thereby demonstrating that sources of within- and between-study heterogeneity and the manner in which data are treated can inhibit the detection of interaction effects in meta-analyses that combine many cohorts with varying characteristics. This suggests that combining many small studies that have measured environmental exposures differently may be relatively inefficient for the detection of gene × environment interactions.
PMCID: PMC3723486  PMID: 23935507
10.  Cognitive Function in Adult Offspring of Women with Gestational Diabetes–The Role of Glucose and Other Factors 
PLoS ONE  2013;8(6):e67107.
We aimed to evaluate cognitive function in adult offspring of women with diet-treated gestational diabetes and to study potential associations with maternal glucose values.
Materials and Methods
In 2003–2005 cognitive function was assessed in a cohort of 18–27 year old offspring of women with diet-treated gestational diabetes mellitus (n = 153) and offspring from the background population (n = 118). The main outcome measure was global cognitive score derived from Raven’s Progressive Matrices and three verbal subtests from the Weschler Adult Intelligence Scale. Maternal fasting- and 2-hour blood glucose values from the diagnostic oral glucose tolerance test were used as exposure variables.
Offspring of women with gestational diabetes mellitus had a lower global cognitive score, than offspring from the background population (93.1 vs. 100.0, P<0.001). However, when adjusted for maternal age at delivery, parity, smoking during pregnancy, pre-pregnancy overweight, family social class, parental educational level, gender, birth weight, gestational age, perinatal complications and offspring age at follow-up, the difference was no longer statistically significant. Offspring global cognitive score decreased significantly with increasing maternal fasting glucose (β = −4.5, 95% CI −8.0 to −0.9, P = 0.01) and 2-hour glucose (β = −1.5, −2.9 to −0.2, P = 0.03) in univariate general linear models, but not when adjusted for family social class and parental educational level.
Lower cognitive test scores in adult offspring of women with diet-treated gestational diabetes were explained by well known predictors of cognitive function, but not by maternal hyperglycaemia during pregnancy. We find it reassuring that mild intrauterine hyperglycaemia does not seem to have adverse effect on offspring cognitive function.
PMCID: PMC3695979  PMID: 23840595
11.  Correction: Does DNA Methylation of PPARGC1A Influence Insulin Action in First Degree Relatives of Patients with Type 2 Diabetes? 
PLoS ONE  2013;8(6):10.1371/annotation/5c3cf392-57b5-4e80-9a66-4997d10200ae.
PMCID: PMC3731442
12.  Genetic Architecture of Vitamin B12 and Folate Levels Uncovered Applying Deeply Sequenced Large Datasets 
PLoS Genetics  2013;9(6):e1003530.
Genome-wide association studies have mainly relied on common HapMap sequence variations. Recently, sequencing approaches have allowed analysis of low frequency and rare variants in conjunction with common variants, thereby improving the search for functional variants and thus the understanding of the underlying biology of human traits and diseases. Here, we used a large Icelandic whole genome sequence dataset combined with Danish exome sequence data to gain insight into the genetic architecture of serum levels of vitamin B12 (B12) and folate. Up to 22.9 million sequence variants were analyzed in combined samples of 45,576 and 37,341 individuals with serum B12 and folate measurements, respectively. We found six novel loci associating with serum B12 (CD320, TCN2, ABCD4, MMAA, MMACHC) or folate levels (FOLR3) and confirmed seven loci for these traits (TCN1, FUT6, FUT2, CUBN, CLYBL, MUT, MTHFR). Conditional analyses established that four loci contain additional independent signals. Interestingly, 13 of the 18 identified variants were coding and 11 of the 13 target genes have known functions related to B12 and folate pathways. Contrary to epidemiological studies we did not find consistent association of the variants with cardiovascular diseases, cancers or Alzheimer's disease although some variants demonstrated pleiotropic effects. Although to some degree impeded by low statistical power for some of these conditions, these data suggest that sequence variants that contribute to the population diversity in serum B12 or folate levels do not modify the risk of developing these conditions. Yet, the study demonstrates the value of combining whole genome and exome sequencing approaches to ascertain the genetic and molecular architectures underlying quantitative trait associations.
Author Summary
Genome-wide association studies have in recent years revealed a wealth of common variants associated with common diseases and phenotypes. We took advantage of the advances in sequencing technologies to study the association of low frequency and rare variants in conjunction with common variants with serum levels of vitamin B12 (B12) and folate in Icelanders and Danes. We found 18 independent signals in 13 loci associated with serum B12 or folate levels. Interestingly, 13 of the 18 identified variants are coding and 11 of the 13 target genes have known functions related to B12 and folate pathways. These data indicate that the target genes at all of the loci have been identified. Epidemiological studies have shown a relationship between serum B12 and folate levels and the risk of cardiovascular diseases, cancers, and Alzheimer's disease. We investigated association between the identified variants and these diseases but did not find consistent association.
PMCID: PMC3674994  PMID: 23754956
14.  Does DNA Methylation of PPARGC1A Influence Insulin Action in First Degree Relatives of Patients with Type 2 Diabetes? 
PLoS ONE  2013;8(3):e58384.
Epigenetics may play a role in the pathophysiology of type 2 diabetes (T2D), and increased DNA methylation of the metabolic master regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) has been reported in muscle and pancreatic islets from T2D patients and in muscle from individuals at risk of T2D. This study aimed to investigate DNA promoter methylation and gene expression of PPARGC1A in skeletal muscle from first degree relatives (FDR) of T2D patients, and to determine the association with insulin action as well as the influence of family relation. We included 124 Danish FDR of T2D patients from 46 different families. Skeletal muscle biopsies were excised from vastus lateralis and insulin action was assessed by oral glucose tolerance tests. DNA methylation and mRNA expression levels were measured using bisulfite sequencing and quantitative real-time PCR, respectively. The average PPARGC1A methylation at four CpG sites situated 867-624 bp from the transcription start was associated with whole-body insulin sensitivity in a paradoxical positive manner (β = 0.12, P = 0.03), supported by a borderline significant inverse correlation with fasting insulin levels (β = −0.88, P = 0.06). Excluding individuals with prediabetes and overt diabetes did not affect the overall result. DNA promoter methylation was not associated with PPARGC1A gene expression. The familiality estimate of PPARGC1A gene expression was high (h2 = 79±27% (h2±SE), P = 0.002), suggesting genetic regulation to play a role. No significant effect of familiality on DNA methylation was found. Taken together, increased DNA methylation of the PPARGC1A promoter is unlikely to play a major causal role for the development of insulin resistance in FDR of patients with T2D.
PMCID: PMC3591301  PMID: 23505498
15.  What Is the Contribution of Two Genetic Variants Regulating VEGF Levels to Type 2 Diabetes Risk and to Microvascular Complications? 
PLoS ONE  2013;8(2):e55921.
Vascular endothelial growth factor (VEGF) is a key chemokine involved in tissue growth and organ repair processes, particularly angiogenesis. Elevated circulating VEGF levels are believed to play a role in type 2 diabetes (T2D) microvascular complications, especially diabetic retinopathy. Recently, a genome-wide association study identified two common single nucleotide polymorphisms (SNPs; rs6921438 and rs10738760) explaining nearly half of the variance in circulating VEGF levels. Considering the putative contribution of VEGF to T2D and its complications, we aimed to assess the effect of these VEGF-related SNPs on the risk of T2D, nephropathy and retinopathy, as well as on variation in related traits.
SNPs were genotyped in several case-control studies: French and Danish T2D studies (Ncases = 6,920-Ncontrols = 3,875 and Ncases = 3,561-Ncontrols = 2,623; respectively), two French studies one for diabetic nephropathy (Ncases = 1,242-Ncontrols = 860) and the other for diabetic retinopathy (Ncases = 1,336-Ncontrols = 1,231). The effects of each SNP on quantitative traits were analyzed in a French general population-based cohort (N = 4,760) and two French T2D studies (N = 3,480). SNP associations were assessed using logistic or linear regressions.
In the French population, we found an association between the G-allele of rs6921438, shown to increase circulating VEGF levels, and increased T2D risk (OR = 1.15; P = 3.7×10−5). Furthermore, the same allele was associated with higher glycated hemoglobin levels (β = 0.02%; P = 9.2×10−3). However, these findings were not confirmed in the Danes. Conversely, the SNP rs10738760 was not associated with T2D in the French or Danish populations. Despite having adequate statistical power, we did not find any significant effects of rs6921438 or rs10738760 on diabetic microvascular complications or the variation in related traits in T2D patients.
In spite of their impact on the variance in circulating VEGF, we did not find any association between SNPs rs6921438 and rs10738760, and the risk of T2D, diabetic nephropathy or retinopathy. The link between VEGF and T2D and its complications might be indirect and more complex than expected.
PMCID: PMC3566098  PMID: 23405237
16.  Equally good fixation of cemented and uncemented cups in total trapeziometacarpal joint prostheses 
Acta Orthopaedica  2013;84(1):98-105.
Background and purpose
Cup failure is a recognized problem in total trapeziometacarpal (TM) joint prostheses; it may be related to poor fixation, which can be revealed by radiostereometric analysis (RSA). We compared the early implant migration of cemented trapezium cups to that of uncemented screw cups.
Patients and methods
In a prospective, parallel-group, randomized patient-blinded clinical trial, we included 32 hands in 28 patients (5 males) with a mean age of 58 (40–77) years and with Eaton stage-2 or -3 osteoarthritis of the trapeziometacarpal joint. Patients were randomized to surgery with a cemented DLC all-polyethylene cup (C) (n = 16) or an uncemented hydroxyapatite-coated chrome-cobalt Elektra screw cup (UC) (n = 16). Uncemented cups were inserted without threading of the bone. Stereoradiographs for evaluation of cup migration (primary effect size) and DASH and pain scores were obtained during 2 years of follow-up.
The 2-year total translation (TT) was similar (p = 0.2): 0.24 mm (SD 0.10) for the C (n = 11) and 0.19 mm (SD 0.16) for the UC (n = 11). Variances were similar (p = 0.4). Judged by RSA, 2 UC cups and 1 C cup became loose (TT > 1 mm). Both UC cups were found to be loose at revision. Grip strength, pain, and DASH scores were similar between groups at all measurement points.
Early implant fixation and clinical outcome were equally good with both cup designs. This is the first clinical RSA study on trapezium cups, and the method appears to be clinically useful for detection of loose implants.
PMCID: PMC3584612  PMID: 23343372
17.  Mechanism-based population modelling for assessment of L-cell function based on total GLP-1 response following an oral glucose tolerance test 
GLP-1 is an insulinotropic hormone that synergistically with glucose gives rise to an increased insulin response. Its secretion is increased following a meal and it is thus of interest to describe the secretion of this hormone following an oral glucose tolerance test (OGTT). The aim of this study was to build a mechanism-based population model that describes the time course of total GLP-1 and provides indices for capability of secretion in each subject. The goal was thus to model the secretion of GLP-1, and not its effect on insulin production. Single 75 g doses of glucose were administered orally to a mixed group of subjects ranging from healthy volunteers to patients with type 2 diabetes (T2D). Glucose, insulin, and total GLP-1 concentrations were measured. Prior population data analysis on measurements of glucose and insulin were performed in order to estimate the glucose absorption rate. The individual estimates of absorption rate constants were used in the model for GLP-1 secretion. Estimation of parameters was performed using the FOCE method with interaction implemented in NONMEM VI. The final transit/indirect-response model obtained for GLP-1 production following an OGTT included two stimulation components (fast, slow) for the zero-order production rate. The fast stimulation was estimated to be faster than the glucose absorption rate, supporting the presence of a proximal–distal loop for fast secretion from L-cells. The fast component (st3 = 8.64·10−5 [mg−1]) was estimated to peak around 25 min after glucose ingestion, whereas the slower component (st4 = 26.2·10−5 [mg−1]) was estimated to peak around 100 min. Elimination of total GLP-1 was characterised by a first-order loss. The individual values of the early phase GLP-1 secretion parameter (st3) were correlated (r = 0.52) with the AUC(0–60 min.) for GLP-1. A mechanistic population model was successfully developed to describe total GLP-1 concentrations over time observed after an OGTT. The model provides indices related to different mechanisms of subject abilities to secrete GLP-1. The model provides a good basis to study influence of different demographic factors on these components, presented mainly by indices of the fast- and slow phases of GLP-1 response.
PMCID: PMC3407884  PMID: 21922329
GLP-1; L-cells; Oral glucose tolerance test (OGTT); Indirect response model; NONMEM
18.  Genetic Variant SCL2A2 Is Associated with Risk of Cardiovascular Disease – Assessing the Individual and Cumulative Effect of 46 Type 2 Diabetes Related Genetic Variants 
PLoS ONE  2012;7(11):e50418.
To assess the individual and combined effect of 46 type 2 diabetes related risk alleles on incidence of a composite CVD endpoint.
Data from the first Danish MONICA study (N = 3523) and the Inter99 study (N = 6049) was used. Using Cox proportional hazard regression the individual effect of each risk allele on incident CVD was analyzed. Risk was presented as hazard ratios (HR) per risk allele.
During 80,859 person years 1441 incident cases of CVD (fatal and non-fatal) occurred in the MONICA study. In Inter99 942 incident cases were observed during 61,239 person years.
In the Danish MONICA study four gene variants were significantly associated with incident CVD independently of known diabetes status at baseline; SLC2A2 rs11920090 (HR 1.147, 95% CI 1.027–1.283 , P = 0.0154), C2CD4A rs7172432 (1.112, 1.027–1.205 , P = 0.0089), GCKR rs780094 (1.094, 1.007–1.188 , P = 0.0335) and C2CD4B rs11071657 (1.092, 1.007–1.183 , P = 0.0323). The genetic score was significantly associated with increased risk of CVD (1.025, 1.010–1.041, P = 0.0016). In Inter99 two gene variants were associated with risk of CVD independently of diabetes; SLC2A2 (HR 1.180, 95% CI 1.038–1.341 P = 0.0116) and FTO (0.909, 0.827–0.998, P = 0.0463). Analysing the two populations together we found SLC2A2 rs11920090 (HR 1.164, 95% CI 1.070–1.267, P = 0.0004) meeting the Bonferroni corrected threshold for significance. GCKR rs780094 (1.076, 1.010–1.146, P = 0.0229), C2CD4B rs11071657 (1.067, 1.003–1.135, P = 0.0385) and NOTCH2 rs10923931 (1.104 (1.001 ; 1.217 , P = 0.0481) were found associated with CVD without meeting the corrected threshold. The genetic score was significantly associated with increased risk of CVD (1.018, 1.006–1.031, P = 0.0043).
This study showed that out of the 46 genetic variants examined only the minor risk allele of SLC2A2 rs11920090 was significantly (P = 0.0005) associated with a composite endpoint of incident CVD below the threshold for statistical significance corrected for multiple testing. This potential pathway needs further exploration.
PMCID: PMC3503928  PMID: 23185617
19.  The Danish Centre for Strategic Research in Type 2 Diabetes (DD2) Project: rationale and planned nationwide studies of genetic predictors, physical exercise, and individualized pharmacological treatment 
Clinical Epidemiology  2012;4(Suppl 1):7-13.
Here we provide an overview of the rationale and methods of a series of planned population based studies within the Danish Centre for Strategic Research in Type 2 Diabetes (DD2) Project. The project aims to support and evaluate ongoing political and administrative efforts to implement nationwide guidelines for maintaining metabolic control in newly diagnosed type 2 diabetes (T2D) patients to prevent diabetic complications and improve quality of life. The DD2 is designed as a prospective cohort study (collection of epidemiological data) supplemented by randomized clinical intervention trials (on physical exercise and individualized pharmacological treatment) and the establishment of a biobank comprised of material from a large number of newly diagnosed T2D patients. Inclusion of the majority of newly diagnosed T2D patients as they are diagnosed at their general practitioner or diabetes hospital outpatient clinics and entered into the DD2 cohort will establish a nationwide database comprising a large number of future incident cases of T2D in Denmark. These cases will form the project cohort of the DD2. Within the first 6 months of diagnosis, all patients will be invited to contribute to a biobank of DNA, plasma, urine, and tissue sampling. The DNA biobank will enable future studies of the effect of pharmacological treatment and outcome in subsets of patients with specific genetic risk profiles covering disease etiology and specific drug kinetics and metabolism. We will also perform two clinical intervention trials examining: the effectiveness of physical exercise on diabetes-related outcomes and the impact of trial outcomes on individualized pharmacological treatment. Moreover, the DD2 will serve as a platform for testing and developing new antidiabetic drugs. All together, we expect this study to contribute to substantially improved diabetes care in T2D patients locally and abroad.
PMCID: PMC3469285  PMID: 23071406
type 2 diabetes; prognosis; intervention; physical exercise
21.  TFAP2B Influences the Effect of Dietary Fat on Weight Loss under Energy Restriction 
PLoS ONE  2012;7(8):e43212.
Numerous gene loci are related to single measures of body weight and shape. We investigated if 55 SNPs previously associated with BMI or waist measures, modify the effects of fat intake on weight loss and waist reduction under energy restriction.
Methods and Findings
Randomized controlled trial of 771 obese adults. (Registration: ISRCTN25867281.) One SNP was selected for replication in another weight loss intervention study of 934 obese adults. The original trial was a 10-week 600 kcal/d energy-deficient diet with energy percentage from fat (fat%) in range of 20–25 or 40–45. The replication study used an 8-weeks diet of 880 kcal/d and 20 fat%; change in fat% intake was used for estimation of interaction effects. The main outcomes were intervention weight loss and waist reduction. In the trial, mean change in fat% intake was −12/+4 in the low/high-fat groups. In the replication study, it was −23/−12 among those reducing fat% more/less than the median. TFAP2B-rs987237 genotype AA was associated with 1.0 kg (95% CI, 0.4; 1.6) greater weight loss on the low-fat, and GG genotype with 2.6 kg (1.1; 4.1) greater weight loss on the high-fat (interaction p-value; p = 0.00007). The replication study showed a similar (non-significant) interaction pattern. Waist reduction results generally were similar. Study-strengths include (i) the discovery study randomised trial design combined with the replication opportunity (ii) the strict dietary intake control in both studies (iii) the large sample sizes of both studies. Limitations are (i) the low minor allele frequency of the TFAP2B polymorphism, making it hard to investigate non-additive genetic effects (ii) the different interventions preventing identical replication-discovery study designs (iii) some missing data for non-completers and dietary intake. No adverse effects/outcomes or side-effects were observed.
Under energy restriction, TFAP2B may modify the effect of dietary fat intake on weight loss and waist reduction.
PMCID: PMC3428346  PMID: 22952648
22.  The PNPLA3 rs738409 G-Allele Associates with Reduced Fasting Serum Triglyceride and Serum Cholesterol in Danes with Impaired Glucose Regulation 
PLoS ONE  2012;7(7):e40376.
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.
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 (ntotal = 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).
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.
Our findings suggest that the G-allele of PNPLA3 rs738409 associates with reduced fasting levels of cholesterol and triglyceride in individuals with IGR.
PMCID: PMC3390392  PMID: 22792295
23.  The predictive value of microRNA-126 in relation to first line treatment with capecitabine and oxaliplatin in patients with metastatic colorectal cancer 
BMC Cancer  2012;12:83.
MicroRNA-126 is the only microRNA (miRNA) known to be endothelial cell-specific influencing angiogenesis in several ways. The aim of the present study was to analyse the possible predictive value of miRNA-126 in relation to first line capecitabine and oxaliplatin (XELOX) in patients with metastatic colorectal cancer (mCRC).
The study included 89 patients with mCRC. In situ hybridization (ISH) was performed to detect miRNA-126 in formalin-fixed paraffin embedded tissue from primary tumours. The expression of miRNA-126, area per image (μm2), was measured using image analysis. Clinical response was evaluated according to RECIST. Progression free survival (PFS) was compared using the Kaplan-Meier method and the log rank test. Tumours were classified as low or high miRNA-126 expressing tumours using the median value from the patients with response as cut-off.
The median miRNA-126 expression level was significantly higher in patients responding to XELOX, 3629 μm2 (95% CI, 2566-4846), compared to the patients not responding, 1670 μm2 (95% CI, 1436-2041), p < 0.0001. The positive predictive value was 90%, and the negative predictive value was 71%. The median PFS of patients with high expressing tumours was 11.5 months (95% CI, 9.0-12.7 months) compared to 6.0 months (95% CI, 4.8-6.9 months) for patients with low expressing tumours, p < 0.0001.
Angiogenesis quantified by ISH of miRNA-126 was related to response to first line XELOX in patients with mCRC, translating to a significant difference in PFS. The predictive value of miRNA-126 remains to be further elucidated in prospective studies.
PMCID: PMC3311029  PMID: 22397399
Angiogenesis; Chemotherapy; Colorectal neoplasms; microRNAs; Predictive biomarkers
24.  The effect of FOXA2 rs1209523 on glucose-related phenotypes and risk of type 2 diabetes in Danish individuals 
BMC Medical Genetics  2012;13:10.
Variations within the FOXA family have been studied for a putative contribution to the risk of type 2 diabetes (T2D), and recently the minor T-allele of FOXA2 rs1209523 was reported to associate with decreased fasting plasma glucose levels in a study using a weighted false discovery rate control procedure to enhance the statistical power of genome wide association studies in detecting associations between low-frequency variants and a given trait.
Thus, the primary aim of this study was to investigate whether the minor T-allele of rs1205923 in FOXA2 associated with 1) decreased fasting plasma glucose and 2) a lower risk of developing T2D. Secondly, we investigated whether rs1205923 in FOXA2 associated with other glucose-related phenotypes.
The variant was genotyped in Danish individuals from four different study populations using KASPar® PCR SNP genotyping system. We examined for associations of the FOXA2 genotype with fasting plasma glucose and estimates of insulin release and insulin sensitivity following an oral glucose tolerance test in 6,162 Danish individuals from the population-based Inter99 study while association with T2D risk was assessed in 10,196 Danish individuals including four different study populations.
The FOXA2 rs1209523 was not associated with fasting plasma glucose (effect size (β) = -0.03 mmol/l (95%CI: -0.07; 0.01), p = 0.2) in glucose-tolerant individuals from the general Danish population. Furthermore, when employing a case-control setting the variant showed no association with T2D (odds ratio (OR) = 0.82 (95%CI: 0.62-1.07), p = 0.1) among Danish individuals. However, when we performed the analysis in a subset of 6,022 non-obese individuals (BMI < 30 kg/m2) an association with T2D was observed (OR = 0.68 (95%CI: 0.49-0.94), p = 0.02). Also, several indices of insulin release and β-cell function were associated with the minor T-allele of FOXA2 rs1209523 in non-obese individuals.
We failed to replicate association of the minor T-allele of FOXA2 rs1209523 with fasting plasma glucose in a population based sample of glucose tolerant individuals. More extensive studies are needed in order to fully elucidate the potential role of FOXA2 in glucose homeostasis.
PMCID: PMC3344680  PMID: 22325233
25.  Early recovery after fast-track Oxford unicompartmental knee arthroplasty 
Acta Orthopaedica  2012;83(1):41-45.
Background and purpose
After total knee arthroplasty with conventional surgical approach, more than half of the quadriceps extension strength is lost in the first postoperative month. Unicompartmental knee arthroplasty (UKA) operated with minimally invasive surgery (MIS) results in less operative trauma. We investigated changes in leg-extension power (LEP) in the first month after MIS Oxford UKA and its relation to pain, knee motion, functional performance, and knee function.
Patients and methods
In 35 consecutive Oxford UKA patients, LEP was measured 1 week before and 1 month after surgery together with knee motion, knee swelling, the 30-second chair-stand test, and Oxford knee score. Assessment of knee pain at rest and walking was done using a visual analog scale.
30 patients were discharged on the day after surgery, and 5 on the second day after surgery. LEP and functional performance reached the preoperative level after 1 month. Only slight postoperative knee swelling was observed with rapid restoration of knee flexion and function. A high level of pain during the first postoperative night and day fell considerably thereafter. None of the patients needed physiotherapy supervision in the first month after discharge.
Fast-track MIS Oxford UKA with discharge on the day after surgery is safe and leads to early recovery of knee motion and strength even when no physiotherapy is used.
PMCID: PMC3278656  PMID: 22313368

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