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The congenital dysfibrinogenemias, most often associated with bleeding disorders, encompass mutations in the amino-terminal end of fibrinogen α-chain consisting of Gly17-Pro18-Arg19-Val20, known as knob A, which is a critical site for fibrin polymerization. Here we review the studies reporting dysfibrinogenemia due to mutations affecting fibrinogen knob A and identified 29 papers. The number of reports on dysfibrinogenemias related to residues Gly17, Pro18, Arg19, and Val20 is 5, 4, 18, and 2, respectively. Dysfibrinogenemias related to residues Gly17, Pro18, and Val20 are exclusively associated with bleeding tendency. However, the clinical picture associated with dysfibrinogenemia related to residue Arg19 varies, with most patients suffering from bleeding tendencies, but also transitory ischemic attacks and retinal thrombosis may occur. The reason for this variation is unclear. To elaborate the genotype–phenotype associations further, we studied a Danish family with knob A-related dysfibrinogenemia caused by the Aα Arg19Gly (p.Arg19Gly) mutation using whole-exome sequencing and fibrin structure analysis. Our family is the first reported carrying the p.Arg19Gly mutation combined with one or more single nucleotide polymorphisms (SNP)s in FGA, FGB, and/or FGG and increased fibrin fiber thickness and fibrin mass-to-length ratio suffering from pulmonary emboli, suggesting that compound genotypes may contribute to the thrombogenic phenotype of these patients. Our review, accordingly, focuses on significance of SNPs, compound genotypes, and fibrin structure measures affecting the genotype–phenotype associations in fibrinogen knob A mutations.

Purpose: Definitive diagnosis of prostate cancer is based on biopsies, a procedure associated with side-effects. The use of biomarkers in blood and urine could potentially help clinicians select patients for whom biopsies are needed. The aim of the study was to test a new urine and plasma biomarker test in detecting medium and high grade prostate cancer. Materials and methods: Blood and urine samples were prospectively collected from 41 patients prior to prostate biopsy or TUR-P and again after 3 months. The cohort included patients with suspicion of prostate cancer and patients with prior prostate cancer diagnosis. The mRNA expression of ten selected genes measured by PCR were used together with clinical data in multiple algorithms for prediction of medium-high grade prostate cancer in prostate biopsies. The testing was originally developed and validated in the USA. The method was transferred to a local Danish laboratory. Medium and high grade cancer was defined as Gleason score ≥ 3 + 4. Results: Using the biomarker test, prior to any prostate procedures, the sensitivity for detecting medium-high grade prostate cancer was 100% and the specificity was 56% and 63%, depending on the cut-off point used. When using the biomarker test, following biopsy or TUR-P, the sensitivity and specificity were reduced to 89% and 28–34% respectively. When comparing results, there was a significant difference (p < 0.05), favoring the test performed prior to the procedures. Conclusions: We were able to predict the presence of medium-high grade prostate cancer, thereby confirming earlier findings of the biomarker test.

Background: Statins treat hyperlipidemia and prevent cardiovascular morbidity and mortality. Evidence suggests that they also have anti-neoplastic activity. Several studies show a reduced rate of breast cancer recurrence among lipophilic statin users (e.g., simvastatin), motivating calls for clinical trials of statins in breast cancer patients. We measured the impact of genetic variation in statin-metabolizing enzymes and drug transporters on the recurrence rate in simvastatin-treated breast cancer patients. Methods: We conducted a nested case-control study among Danish women diagnosed with non-metastatic, invasive breast cancer between 2004–2010 who had filled ≥1 prescription for simvastatin after diagnosis. Cases were all breast cancer recurrences from the source population; one control was matched to each case on cancer stage, estrogen receptor and hormone therapy status, calendar period of diagnosis, and duration of simvastatin exposure. We genotyped variants in simvastatin-metabolizing enzymes (CYP3A4/rs35599367 and CYP3A5/rs776746) and drug transporters (ABCB1/rs2032582 and SLCO1B1/rs4149056), and estimated their association with recurrence with logistic regression models. Results: We observed protective (though imprecisely-measured) associations between variants in genes encoding drug transporters (ABCB1 and SLCO1B1) and simvastatin-metabolizing enzymes (CYP3A4 and CYP3A5) and breast cancer recurrence in simvastatin-treated women. For example, carrying two variant alleles in ABCB1 was associated with a 31% lower rate of recurrence (multivariable OR = 0.69, 95% CI: 0.31, 1.5). Conclusion: Our study provides weak evidence to support the use of genetic variation in ABCB1, SLCO1B1, CYP3A4, and CYP3A5 as biomarkers of breast tumor response to simvastatin. Validation of these findings within adjuvant clinical trials is encouraged.

AIMS: The present study tested the hypothesis that metformin treatment may increase myocardial efficiency (stroke work/myocardial oxygen consumption) in insulin-resistant patients with heart failure and reduced ejection fraction (HFrEF) without diabetes.METHODS AND RESULTS: Thirty-six HFrEF patients (ejection fraction 37 ± 8%; median age 66 years) were randomised to metformin (n = 19) or placebo (n = 17) for 3 months in addition to standard heart failure therapy. The primary endpoint was change in myocardial efficiency expressed as the work metabolic index (WMI), assessed by 11 C-acetate positron emission tomography and transthoracic echocardiography. Compared with placebo, metformin treatment (1450 ± 550 mg/day) increased WMI [absolute mean difference, 1.0 mmHg·mL·m-2 ·106 ; 95% confidence interval (CI) 0.1 to 1.8; P = 0.03], equivalent to a 20% relative efficiency increase. Patients with above-median plasma metformin levels displayed greater WMI increase (25% vs. -4%; P = 0.02). Metformin reduced myocardial oxygen consumption (-1.6 mL O2 ·100 g-1 ·min-1 ; P = 0.014). Cardiac stroke work was preserved (-2 J; 95% CI -11 to 7; P = 0.69). Metformin reduced body weight (-2.2 kg; 95% CI -3.6 to -0.8; P = 0.003) and glycated haemoglobin levels (-0.2%; 95% CI -0.3 to 0.0; P = 0.02). Changes in resting and exercise ejection fraction, global longitudinal strain, and exercise capacity did not differ between groups.CONCLUSION: Metformin treatment in non-diabetic HFrEF patients improved myocardial efficiency by reducing myocardial oxygen consumption. Measurement of circulating metformin levels differentiated responders from non-responders. These energy-sparing effects of metformin encourage further large-scale investigations in heart failure patients without diabetes.

Background Type 2 diabetes mellitus (T2DM) is an independent risk factor of cardiovascular disease (CVD), however, the underlying mechanisms are largely unknown. Using non-atherosclerotic internal thoracic arteries (ITAs) obtained from coronary artery bypass grafting, we previously identified a distinct elevation in the level of proteins comprising the arterial basement membrane in T2DM patients not treated with metformin. Altered transcription of genes encoding these proteins has not been observed, indicating alternative mechanisms of dysregulation. Methods In this study we screened for differential expression of arterial microRNAs (miRNAs) in T2DM patients to test the hypothesis that the arterial protein signature of diabetic patients is associated with dysregulation at the miRNA level, and further to lay the foundation for novel hypotheses addressing the increased CVD risk of T2DM patients. MiRNA isolated from fresh frozen ITAs [from 18 T2DM- (10 of which were subject to metformin treatment) and 30 non-diabetes mellitus (non-DM) patients] were analyzed by microarray, and miRNAs isolated from formalin-fixated paraffin-embedded (FFPE) ITAs were analyzed by quantitative PCR (qPCR) in an independent study group [26 T2DM- (15 of which were subject to metformin treatment) and 26 non-DM patients] to determine expression levels of miRNAs in a pre-defined panel of 12 miRNAs. Results Unexpectedly, no miRNAs were found to be affected by T2DM status in either of the two study groups. Conclusions Our data suggest that alternatives to microRNA dysregulation underlie T2DM-associated protein changes in non-atherosclerotic arteries. Electronic supplementary material The online version of this article (10.1186/s12933-018-0715-y) contains supplementary material, which is available to authorized users.

BACKGROUND: Distinguishing between low- and high-grade prostate cancers (PCa) is important, but biopsy may underestimate the actual grade of cancer. We have previously shown that urine/plasma-based prostate-specific biomarkers can predict high grade PCa. Our objective was to determine the accuracy of a test using cell-free RNA levels of biomarkers in predicting prostatectomy results.METHODS: This multicenter community-based prospective study was conducted using urine/blood samples collected from 306 patients. All recruited patients were treatment-naïve, without metastases, and had been biopsied, designated a Gleason Score (GS) based on biopsy, and assigned to prostatectomy prior to participation in the study. The primary outcome measure was the urine/plasma test accuracy in predicting high grade PCa on prostatectomy compared with biopsy findings. Sensitivity and specificity were calculated using standard formulas, while comparisons between groups were performed using the Wilcoxon Rank Sum, Kruskal-Wallis, Chi-Square, and Fisher's exact test.RESULTS: GS as assigned by standard 10-12 core biopsies was 3 + 3 in 90 (29.4%), 3 + 4 in 122 (39.8%), 4 + 3 in 50 (16.3%), and > 4 + 3 in 44 (14.4%) patients. The urine/plasma assay confirmed a previous validation and was highly accurate in predicting the presence of high-grade PCa (Gleason ≥3 + 4) with sensitivity between 88% and 95% as verified by prostatectomy findings. GS was upgraded after prostatectomy in 27% of patients and downgraded in 12% of patients.CONCLUSIONS: This plasma/urine biomarker test accurately predicts high grade cancer as determined by prostatectomy with a sensitivity at 92-97%, while the sensitivity of core biopsies was 78%.

Background: Unnecessary biopsies and overdiagnosis of prostate cancer (PCa) remain a serious healthcare problem. We have previously shown that urine- and plasma-based prostate-specific biomarkers when combined can predict high grade prostate cancer (PCa). To further validate this test, we performed a prospective multicenter study recruiting patients from community-based practices. Patients and Methods: Urine and plasma samples from 2528 men were tested prospectively. Results were correlated with biopsy findings, if a biopsy was performed as deemed necessary by the practicing urologist. Of the 2528 patients, biopsy was performed on only 524 (21%) patients. Results: Of the 524 patients, Gleason≥3+4 PCa was found in 161 (31%) and Gleason ≥4+3 was found in 62 (12%) of the patients. The urine/plasma biomarkers algorithm showed sensitivity and specificity of 75% and 69% for predicting Gleason ≥3+4. However, upon incorporating prostate size and prior history of biopsy in the algorithm, we achieved a sensitivity between 97% and 86% and a specificity between 36% and 57%, dependent on the used cut-off point. Sensitivity for predicting PCa Gleason ≥4+3 was between 96% and 99% and specificity between 59% and 37%, dependent on the cut-off point. Diagnosis of Gleason ≥3+4 was missed in 1% to 3% of tested patients and of Gleason ≥4+3 in 0.2% to 1%. Conclusion: This test when integrated with prostate volume and the prior prostate biopsy enhance the sensitivity and specificity for predicting the presence of high grade prostate cancer with negative predictive value (NPV) of 90% to 97% for Gleason ≥3+4 and between 98% to 99% for Gleason ≥4+3.

Aims: Metformin is first-line treatment of type 2 diabetes mellitus and reduces cardiovascular events in patients with insulin resistance and type 2 diabetes. Target tissue for metformin action is thought to be the liver, where metformin distribution depends on facilitated transport by polyspecific transmembrane organic cation transporters (OCTs). Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the western world with strong associations to insulin resistance and the metabolic syndrome, but whether NAFLD affects metformin biodistribution to the liver is not known. In this study, the primary aim was to investigate in vivo hepatic uptake of metformin dynamically in humans with variable degrees of liver affection. As a secondary aim, we wished to correlate hepatic metformin distribution with OCT gene transcription determined in diagnostic liver biopsies. Methods: Eighteen patients with biopsy-proven NAFLD were investigated using 11C-metformin PET/CT technique. Gene transcripts of OCTs were determined by real-time polymerase chain reaction (PCR). Results: We observed similar hepatic volume of distribution of metformin between patients with simple steatosis and non-alcoholic steatohepatitis (NASH) (Vd 2.38 ± 0.56 vs. 2.10 ± 0.39, P = 0.3). There was no association between hepatic exposure to metformin and the degree of inflammation or fibrosis, and no clear correlation between metformin distribution and OCT gene transcription. Conclusion: Metformin is distributed to the liver in patients with NAFLD and the distribution is not impaired by inflammation or fibrosis. The findings imply that metformin action in liver in patients with NAFLD may be preserved.

Background Factors such as age, gender and genetic polymorphisms may explain individual difference in pain phenotype. Genetic associations to pain sensitivity have previously been investigated in osteoarthritis patients with focus on the P2X7, TRPV1 and TACR1 genes. However, other genes may play a role as well. Osteoarthritis is a common joint disease and many patients suffering from this disease are thought to have increased sensitivity to noxious stimuli resulting from sensitization in the nociceptive system. The aim of the study was to investigate if genetic variants of mu, kappa, and delta opioid receptor genes (OPRM1, OPRK1, and OPRD1), and the catechol-O-methyltransferase gene (COMT) influenced the pain phenotype in patients with osteoarthritis. Methods The frequencies of seventeen polymorphisms were examined. Pain sensitivity was assessed pre-operatively by: 1) hip rotation; 2) contact heat stimulation; 3) conditioned pain modulation effect and 4) pressure stimulation at the tibia in both the affected and the unaffected leg. Results Ninety-two patients (mean age 66 years) with unilateral hip osteoarthritis were included in the study. Carriage of the OPRM1 rs589046T allele was found to be associated with increased pain rating during hip rotation (P=0.04) and increased conditioned pain modulation (P=0.049). Carriage of the OPRD1 rs2234918C allele was found to be associated with increased pain detection threshold to contact heat stimulation (P=0.001). No other associations were found (all P > 0.05). Conclusion Results from the present study suggest that, in patients with hip osteoarthritis, genetic variants in OPRM1 and OPRD1 may contribute to the pain phenotype. This article is protected by copyright. All rights reserved.

Neurobiology is regulated by miRNA. Here circulating plasma miRNAs were assayed on a 754 miRNA OpenArray platform using 90 monozygotic elderly twins (73–95 year of age) and associated with mini mental state examination (MMSE) and a five-component cognitive score (CCS) in an explorative study. Both ordinary individual and twin-pair analyses were performed with level of cognitive scores. Candidate miRNAs were further associated with cognitive decline over 10 years using up to six repeated assessments. A total of 278 miRNAs were expressed in plasma from at least ten participants and 23 miRNAs were nominally associated (i.e., at an uncorrected p < 0.05) with CCS or MMSE in the paired analyses. Generally, elderly individuals with poor cognitive function had increase miRNA expression compared with equivalent individuals who performed better on the cognitive scale. Three miRNAs, miR-151a-3p, miR-212-3p and miR-1274b were associated with CCS both in the paired and the individual analysis. Four miRNAs found to be associated with CCS in cross-sectional analysis were also found to show an association in longitudinal analysis such that increase miRNA expression was associated with steeper cognitive decline. We propose a shared biological path underlies dementia and normative cognitive aging.

Background. Docetaxel is a highly effective treatment of a wide range of malignancies but is often associated with peripheral neuropathy. The genetic variability of genes involved in the transportation or metabolism of docetaxel may be responsible for the variation in docetaxel-induced peripheral neuropathy (DIPN). The main purpose of this study was to investigate the impact of genetic variants in GSTP1 and ABCB1 on DIPN.Material and methods. DNA was extracted from whole blood from 150 patients with early-stage breast cancer who had received adjuvant docetaxel from February 2011 to May 2012. Two polymorphisms in GSTP1 and three in ABCB1 were selected for the primary analysis, and a host of other candidate genes was explored and compared between 75 patients with clinician-reported DIPN grade ≥ 2 and 75 patients without DIPN.Results. Patients with the genetic variants GSTP1 rs1138272 C/T or T/T (114Ala/114Val or 114Val/114Val) genotype had an adjusted odds ratio of 3.82; 95% confidence interval 1.34–11.09 of developing DIPN. This result was confirmed in both analysis of cumulated docetaxel dose and haplotype analysis. None of the explorative genes investigated were significantly correlated with DIPN. Patients with a BMI ≥ 30 were five-fold more likely to have DIPN than patients with BMI < 25.Conclusion. We found that GSTP1 Ala114Val polymorphism is associated with occurrence of DIPN. This supports the theory that oxidative stress is involved in DIPN pathophysiology. If confirmed, this may be helpful in the risk assessment of DIPN and perhaps help to achieve better management of neurotoxicity.

BACKGROUND Glioblastoma (GBM) is the most common and aggressive primary brain tumor having a median overall survival below 15 months. To improve the prognosis, biomarkers are needed for early detection of GBM progression. In the present study we identified differentially expressed microRNAs (miRNAs) in plasma samples from GBM patients and investigated their potential to function as noninvasive biomarkers.

BACKGROUND: Dysfibrinogenemia is a rare group of qualitative fibrinogen disorders caused by structural abnormalities in the fibrinogen molecule. The laboratory diagnosis of dysfibrinogenemia is controversial. Fibrinogen Paris V, clinically termed Dusart Syndrome, is a dysfibrinogenemia caused by a single base substitution in the gene coding for the Aα-chain of the fibrinogen molecule.OBJECTIVES: To diagnose the first Scandinavian family with Fibrinogen Paris V affecting several family members; the proband, a seven-year-old boy with cerebral vein thrombosis.METHODS: The diagnosis was established following the ISTH guideline for laboratory testing supplemented with fibrin structure analysis and fibrinogen gene analysis.RESULTS: Prolonged thrombin time and reduced ratio between the functional and the protein concentration of fibrinogen were observed in four family members who also were characterized by significantly reduced fibrin polymerization (p < 0.001), reduced fibrin fibre diameter (p < 0.001), reduced fibrin mass-length ratio (p < 0.001) and significantly reduced t-PA-induced fibrinolysis of the fibrin clots (p < 0.001) when compared to controls. Fibrinogen gene analysis demonstrated that five of the family members carried the Fibrinogen Paris V mutation. All laboratory tests were normal in the family members carrying the wild type of the fibrinogen gene. Four of the affected patients had suffered from thrombotic episodes. A noticeable feature in the present family was the presence of both venous and arterial thrombosis.CONCLUSIONS: Excellent concordance was observed between the screening and confirmatory tests, fibrin structure analysis and fibrinogen gene analysis. Fibrin structure analysis should be considered in the laboratory algorithm for diagnosis of dysfibrinogenemia.

Familial lipoprotein lipase (LPL) deficiency (FLLD) is a rare autosomal recessive genetic disorder caused by homozygous or compound heterozygous mutations in the LPL gene. FLLD individuals usually express an impaired or non-functional LPL enzyme with low or absent triglyceride (TG) hydrolysis activity causing severe hypertriglyceridaemia. Here we report a case of FLLD in a 29-year-old man, who initially presented with eruptive cutaneous xanthomata, elevated plasma TG concentration but no other co-morbidities. Subsequent genetic testing of the patient revealed compound heterozygosity of a novel duplication (p.R44Kfs*4) leading to a premature stop codon in exon 2 and a known mutation (N291S) in exon 5 of the LPL gene. Further biochemical analysis of the patient's postheparin plasma confirmed a reduction of total lipase activity compared with his heterozygous father carrying the common N291S mutation and to a healthy control. Also the patient showed increased (1.85-fold) activity of hepatic lipase (HL), indicating a functional link between HL and LPL. In summary, we report a case of FLLD caused by compound heterozygosity of a new duplication and a common mutation in the LPL gene, resulting in residual LPL activity. With such mutations, individuals may not receive a diagnosis before classical FLLD symptoms appear later in adulthood. Nevertheless, early diagnosis and lipid-lowering treatment may favour a reduced risk of premature cardiovascular disease or acute pancreatitis in such individuals.

OBJECTIVE: The aim of this study was to examine the effect of single nucleotide polymorphisms in CYP2C8, LPIN1, PPARGC1A and PPARγ on rosiglitazone's (i) trough steady-state plasma concentration (C(ss,min)), (ii) on glycosylated haemoglobin A1c (HbA1c) and (iii) the risk of developing adverse events, mainly oedema, in patients with type 2 diabetes mellitus (T2D).METHODS: The data used in this study were obtained from the South Danish Diabetes Study including 371 T2D patients with a focus on the 187 patients who were treated with rosiglitazone. The study was a placebo-controlled, partly blinded and multicentre clinical trial. The C(ss,min) of rosiglitazone and HbA1c was determined and the genotype of the patients was identified.RESULTS: The mean C(ss,min) of rosiglitazone was 21.3 ng/ml (95% confidence interval 18.8; 24.2 ng/ml), with observations ranging from 1 to 296 ng/ml. Carriers of CYP2C8*3 (n=32) (rs10509681 and rs11572080) had a statistically significantly lower mean C(ss,min) than wild types (n=106), and they also had a statistically significantly lower mean absolute difference in HbA1c during rosiglitazone treatment. Finally, the carriers of CYP2C8*3 had a lower odds ratio of developing oedema.CONCLUSION: We showed that CYP2C8*3 was associated with lower plasma levels of rosiglitazone and hence a reduced therapeutic response but also a lower risk of developing oedema during treatment with rosiglitazone. Individualized treatment with rosiglitazone on the basis of the CYP2C8 genotype may therefore be possible.

Familial hypercholesterolemia (FH) is one of the most frequent single-gene disorders; nevertheless, it is commonly underdiagnosed and undertreated. To increase the number of individuals diagnosed and treated for FH, an ongoing discovery of novel FH mutations is necessary as a prerequisite to implement good nationwide genetic FH screening strategies. Here we report on the finding of a seldom exon 7–12 duplication in the low-density lipoprotein receptor gene of three Danish patients with FH.

GWAS-based association between RWDD3 and TECTA variants and paclitaxel induced neuropathy could not be confirmed in Scandinavian ovarian cancer patients

Brown adipose tissue activity dissipates energy as heat, and there is evidence that lack of the retinoblastoma protein (pRb) may favor the development of the brown adipocyte phenotype in adipose cells. In this work we assessed the impact of germ line haploinsufficiency of the pRb gene (Rb) on the response to high-fat diet feeding in mice. Rb+/−mice had body weight and adiposity indistinguishable from that of wild-type (Rb+/+) littermates when maintained on a standard diet, yet they gained less body weight and body fat after long-term high-fat diet feeding coupled with reduced feed efficiency and increased rectal temperature. Rb haploinsufficiency ameliorated insulin resistance and hepatosteatosis after high-fat diet in male mice, in which these disturbances were more marked than in females. Compared with wild-type littermates, Rb+/−mice fed a high-fat diet displayed higher expression of peroxisome proliferator-activated receptor (PPAR)γ as well as of genes involved in mitochondrial function, cAMP sensitivity, brown adipocyte determination, and tissue vascularization in white adipose tissue depots. Furthermore, Rb+/−mice exhibited signs of enhanced activation of brown adipose tissue and higher expression levels of PPARα in liver and of PPARδ in skeletal muscle, suggestive of an increased capability for fatty acid oxidation in these tissues. These findings support a role for pRb in modulating whole body energy metabolism and the plasticity of the adipose tissues in vivo and constitute first evidence that partial deficiency in the Rb gene protects against the development of obesity and associated metabolic disturbances.