The hepatokine follistatin is elevated in patients with type 2 diabetes (T2D) and promotes hyperglycemia in mice. Here we explore the relationship of plasma follistatin levels with incident T2D and ...mechanisms involved. Adjusted hazard ratio (HR) per standard deviation (SD) increase in follistatin levels for T2D is 1.24 (CI: 1.04-1.47, p < 0.05) during 19-year follow-up (n = 4060, Sweden); and 1.31 (CI: 1.09-1.58, p < 0.01) during 4-year follow-up (n = 883, Finland). High circulating follistatin associates with adipose tissue insulin resistance and non-alcoholic fatty liver disease (n = 210, Germany). In human adipocytes, follistatin dose-dependently increases free fatty acid release. In genome-wide association study (GWAS), variation in the glucokinase regulatory protein gene (GCKR) associates with plasma follistatin levels (n = 4239, Sweden; n = 885, UK, Italy and Sweden) and GCKR regulates follistatin secretion in hepatocytes in vitro. Our findings suggest that GCKR regulates follistatin secretion and that elevated circulating follistatin associates with an increased risk of T2D by inducing adipose tissue insulin resistance.
Background Abdominal aortic aneurysm (AAA) is a vascular disease with a mortality rate of >80% if ruptured. Mitochondrial dysfunction has been previously implicated in AAA pathogenesis. In this ...study, we aimed to characterize the mitochondrial genetic landscape in AAA. Methods and Results Whole mitochondrial genome sequencing and bioinformatics analysis were performed in comorbidity matched 48 cases without AAA and 48 cases with AAA, objectively diagnosed, and selected from a cohort of 65-year-old men recruited for a screening program. We identified differential mutational landscapes in men with and without AAA, with errors in mitochondrial DNA replication or repair as potential sources. Heteroplasmic insertions and overall heteroplasmy of structural rearrangements were significantly elevated in AAA cases. Three heteroplasmic variants were associated with risk factors of AAA: leukocyte concentration, plasma glucose, and cholesterol levels, respectively. Interestingly, mutations were more prevalent in regulatory part of the mitochondria, the displacement loop region, in AAA as compared with controls (
value <0.05), especially in the conserved and critical mitochondrial extended termination-associated sequence region. Moreover, we report a novel 24 bp mitochondrial DNA duplication present exclusively in cases with AAA (4%) and 75% of the unmatched AAA biopsies. Finally, the haplogroup cluster JTU was overrepresented in AAA and significantly associated with a positive family history of AAA (odds ratio, 2.9 95% CI, 1.1-8.1). Conclusions This is the first study investigating the mitochondrial genome in AAA, where important genetic alterations and haplogroups associated with AAA and clinical risk factors were identified. Our findings have the potential to fill in gaps in the missing genetic information on AAA.
Abstract
Background
Breast cancer is, despite screening, not always detected early enough and is together with other tumor types known to shed genetic information in circulation. Unlike single-copy ...nuclear DNA, mitochondrial DNA (mtDNA) copies range from 100s to 10,000s per cell, thus providing a potentially alternative to identify potential missing cancer information in circulation at an early stage.
Methods
To characterize mitochondrial mutation landscapes in breast cancer, whole mtDNA sequencing and bioinformatics analyses were performed on 86 breast cancer biopsies and 50 available matched baseline cancer-free whole blood samples from the same individuals, selected from a cohort of middle-aged women in Sweden. To determine whether the mutations can be detected in blood plasma prior to cancer diagnosis, we further designed a nested case-control study (n = 663) and validated the shortlisted mutations using droplet digital PCR.
Results
We detected different mutation landscapes between biopsies and matched whole blood samples. Compared to whole blood samples, mtDNA from biopsies had higher heteroplasmic mutations in the D-loop region (
P
= 0.02),
RNR2
(
P
= 0.005),
COX1
(
P
= 0.037) and
CYTB
(
P
= 0.006). Furthermore, the germline mtDNA mutations had higher heteroplasmy level than the lost (
P
= 0.002) and de novo mutations (
P
= 0.04). The nonsynonymous to synonymous substitution ratio (dN/dS) was higher for the heteroplasmic mutations (
P
= 7.25 × 10
−12
) than that for the homoplasmic mutations, but the de novo (
P
= 0.06) and lost mutations (
P
= 0.03) had lower dN/dS than the germline mutations. Interestingly, we found that the critical regions for mitochondrial transcription: MT-HSP1 (odds ratio OR: 21.41), MT-TFH (OR: 7.70) and MT-TAS2 (OR: 3.62), had significantly higher heteroplasmic mutations than the rest of the D-loop sub-regions. Finally, we found that the presence of mt.16093T > C mutation increases 67% risk of developing breast cancer.
Conclusions
Our findings show that mitochondrial genetic landscape changes during cancer pathogenesis and positive selection of mtDNA heteroplasmic mutations in breast cancer. Most importantly, the mitochondrial mutations identified in biopsies can be traced back in matched plasma samples and could potentially be used as early breast cancer diagnostic biomarkers.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Background and Objective
Tinnitus would benefit from an objective biomarker. The goal of this study is to identify plasma biomarkers of constant and chronic tinnitus among selected circulating ...inflammatory proteins.
Methods
A case–control retrospective study on 548 cases with constant tinnitus and 548 matched controls from the Swedish Tinnitus Outreach Project (STOP), whose plasma samples were examined using Olink’s Inflammatory panel. Replication and meta-analysis were performed using the same method on samples from the TwinsUK cohort. Participants from LifeGene, whose blood was collected in Stockholm and Umeå, were recruited to STOP for a tinnitus subtyping study. An age and sex matching was performed at the individual level. TwinsUK participants (
n
= 928) were selected based on self-reported tinnitus status over 2 to 10 years. Primary outcomes include normalized levels for 96 circulating proteins, which were used as an index test. No reference standard was available in this study.
Results
After adjustment for age, sex, BMI, smoking, hearing loss, and laboratory site, the top proteins identified were FGF-21, MCP4, GDNF, CXCL9, and MCP-1; however, these were no longer statistically significant after correction for multiple testing. Stratification by sex did not yield any significant associations. Similarly, associations with hearing loss or other tinnitus-related comorbidities such as stress, anxiety, depression, hyperacusis, temporomandibular joint disorders, and headache did not yield any significant associations. Analysis in the TwinsUK failed in replicating the top candidates. Meta-analysis of STOP and TwinsUK did not reveal any significant association. Using elastic net regularization, models exhibited poor predictive capacity tinnitus based on inflammatory markers sensitivity = 0.52 (95% CI 0.47–0.57), specificity = 0.53 (0.48–0.58), positive predictive value = 0.52 (0.47–0.56), negative predictive values = 0.53 (0.49–0.58), and AUC = 0.53 (0.49–0.56).
Discussion
Our results did not identify significant associations of the selected inflammatory proteins with constant tinnitus. Future studies examining longitudinal relations among those with more severe tinnitus and using more recent expanded proteomics platforms and sampling of cerebrospinal fluid could increase the likelihood of identifying relevant molecular biomarkers.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Anastomotic leakage after surgery for colorectal cancer is a serious complication, causing an increased morbidity rate and mortality rate1,2.
There is a debate and conflicting evidence on whether ...non-steroidal anti-inflammatory drugs (NSAIDs) increase the risk of leak3,4. NSAIDs act by inhibiting cyclooxygenase (COX) enzymes, which can be subdivided into isoenzymes COX-1 and COX-2. In a seminal study by Reisinger et al.5, knocking out the COX-2 gene resulted in an increase of colonic anastomotic leaks in mice. In a complementary cohort of colorectal cancer patients5, an increased frequency of anastomotic leaks was demonstrated among those homozygous for the COX-2 gene promoter mutation −765G > C (also known as rs20417). This finding could potentially be translated into clinical use following external validation.
Biological effects might not only be present among those homozygous for the minor allele of −765C/C. For example, the heterozygous state of −765G/C has been associated with a decreased postoperative inflammatory response6.
The present study aimed to evaluate the prevalence of the polymorphism 765G > C in a Swedish cohort of colorectal cancer patients, and its association with postoperative peritoneal infection.
The human plasma proteome is important for many biological processes and targets for diagnostics and therapy. It is therefore of great interest to understand the interplay of genetic and ...environmental factors to determine the specific protein levels in individuals and to gain a deeper insight of the importance of genetic architecture related to the individual variability of plasma levels of proteins during adult life.
We have combined whole-genome sequencing, multiplex plasma protein profiling, and extensive clinical phenotyping in a longitudinal 2-year wellness study of 101 healthy individuals with repeated sampling. Analyses of genetic and non-genetic associations related to the variability of blood levels of proteins in these individuals were performed.
The analyses showed that each individual has a unique protein profile, and we report on the intra-individual as well as inter-individual variation for 794 plasma proteins. A genome-wide association study (GWAS) using 7.3 million genetic variants identified by whole-genome sequencing revealed 144 independent variants across 107 proteins that showed strong association (P < 6 × 10
) between genetics and the inter-individual variability on protein levels. Many proteins not reported before were identified (67 out of 107) with individual plasma level affected by genetics. Our longitudinal analysis further demonstrates that these levels are stable during the 2-year study period. The variability of protein profiles as a consequence of environmental factors was also analyzed with focus on the effects of weight loss and infections.
We show that the adult blood levels of many proteins are determined at birth by genetics, which is important for efforts aimed to understand the relationship between plasma proteome profiles and human biology and disease.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
We developed and implemented software for the analysis of genome-wide association studies in the context of biological pathway enrichment and have here applied our algorithm to the study of Alzheimer ...disease (AD). Using genome-wide association data in a large French population, we observed a highly significant enrichment of genes involved in intracellular protein transmembrane transport, including several mitochondrial proteins and nucleoporins. An intriguing aspect of these findings is the implication that TOMM40, the channel-forming subunit of the translocase of the mitochondrial outer membrane complex, and a gene generally considered to be indiscernible from APOE because of linkage disequilibrium, may itself contribute to Alzheimer pathology. Results provide an indication that protein trafficking, in particular across the nuclear and mitochondrial membranes, may contribute to risk for AD.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Serological testing is essential to curb the consequences of the COVID-19 pandemic. However, most assays are still limited to single analytes and samples collected within healthcare. Thus, we ...establish a multianalyte and multiplexed approach to reliably profile IgG and IgM levels against several versions of SARS-CoV-2 proteins (S, RBD, N) in home-sampled dried blood spots (DBS). We analyse DBS collected during spring of 2020 from 878 random and undiagnosed individuals from the population in Stockholm, Sweden, and use classification approaches to estimate an accumulated seroprevalence of 12.5% (95% CI: 10.3%-14.7%). This includes 5.4% of the samples being IgG
IgM
against several SARS-CoV-2 proteins, as well as 2.1% being IgG
IgM
and 5.0% being IgG
IgM
for the virus' S protein. Subjects classified as IgG
for several SARS-CoV-2 proteins report influenza-like symptoms more frequently than those being IgG
for only the S protein (OR = 6.1; p < 0.001). Among all seropositive cases, 30% are asymptomatic. Our strategy enables an accurate individual-level and multiplexed assessment of antibodies in home-sampled blood, assisting our understanding about the undiagnosed seroprevalence and diversity of the immune response against the coronavirus.
Enhanced by the growing number of biobanks, biomarker studies can now be performed with reasonable statistical power by using large sets of samples. Antibody-based proteomics by means of suspension ...bead arrays offers one attractive approach to analyze serum, plasma, or CSF samples for such studies in microtiter plates. To expand measurements beyond single batches, with either 96 or 384 samples per plate, suitable normalization methods are required to minimize the variation between plates. Here we propose two normalization approaches utilizing MA coordinates. The multidimensional MA (multi-MA) and MA-loess both consider all samples of a microtiter plate per suspension bead array assay and thus do not require any external reference samples. We demonstrate the performance of the two MA normalization methods with data obtained from the analysis of 384 samples including both serum and plasma. Samples were randomized across 96-well sample plates, processed, and analyzed in assay plates, respectively. Using principal component analysis (PCA), we could show that plate-wise clusters found in the first two components were eliminated by multi-MA normalization as compared with other normalization methods. Furthermore, we studied the correlation profiles between random pairs of antibodies and found that both MA normalization methods substantially reduced the inflated correlation introduced by plate effects. Normalization approaches using multi-MA and MA-loess minimized batch effects arising from the analysis of several assay plates with antibody suspension bead arrays. In a simulated biomarker study, multi-MA restored associations lost due to plate effects. Our normalization approaches, which are available as R package MDimNormn, could also be useful in studies using other types of high-throughput assay data.
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IJS, KILJ, NUK, PNG, UL, UM
Mammographic breast density is one of the strongest risk factors for breast cancer, but molecular understanding of how breast density relates to cancer risk is less complete. Studies of proteins in ...blood plasma, possibly associated with mammographic density, are well-suited as these allow large-scale analyses and might shed light on the association between breast cancer and breast density.
Plasma samples from 1329 women in the Swedish KARMA project, without prior history of breast cancer, were profiled with antibody suspension bead array (SBA) assays. Two sample sets comprising 729 and 600 women were screened by two different SBAs targeting a total number of 357 proteins. Protein targets were selected through searching the literature, for either being related to breast cancer or for being linked to the extracellular matrix. Association between proteins and absolute area-based breast density (AD) was assessed by quantile regression, adjusting for age and body mass index (BMI).
Plasma profiling revealed linear association between 20 proteins and AD, concordant in the two sets of samples (p < 0.05). Plasma levels of seven proteins were positively associated and 13 proteins negatively associated with AD. For eleven of these proteins evidence for gene expression in breast tissue existed. Among these, ABCC11, TNFRSF10D, F11R and ERRF were positively associated with AD, and SHC1, CFLAR, ACOX2, ITGB6, RASSF1, FANCD2 and IRX5 were negatively associated with AD.
Screening proteins in plasma indicates associations between breast density and processes of tissue homeostasis, DNA repair, cancer development and/or progression in breast cancer. Further validation and follow-up studies of the shortlisted protein candidates in independent cohorts will be needed to infer their role in breast density and its progression in premenopausal and postmenopausal women.
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