Publications by authors named "Peter H Nissen"

49 Publications

Impact of centrifugation time and pneumatic tube transport on plasma concentrations of direct oral anticoagulants.

Int J Lab Hematol 2021 Oct 12. Epub 2021 Oct 12.

Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.

Introduction: Rapid results are needed when plasma concentrations of direct oral anticoagulants (DOACs) are required in acute clinical settings. We evaluated the impact of centrifugation time and pneumatic tube transport on DOAC plasma concentrations with the overall aim of reducing turnaround time.

Methods: Blood samples were spiked with rivaroxaban, apixaban or dabigatran in a low and a high concentration prior to centrifugation for 25 minutes (3163 g) or 5 minutes (3000 g) (n = 20 for each DOAC). Both samples spiked with DOACs (n = 20 for each DOAC) and patient samples (n = 25 in total) were transported manually or by pneumatic tube system samples.

Results: For samples spiked with DOAC, statistically significant differences in DOAC plasma concentrations were found between centrifugation times for rivaroxaban in low (P < .05) and high (P < .05) concentrations. Relative bias was below 9% for all DOACs. Statistically significant differences were found between modes of transportation for rivaroxaban (P < .01) and dabigatran (P < .01) in high concentrations. Relative bias was 4%-23% for all DOACs. For patient samples, no statistically significant differences were found between modes of transportation, and relative bias was below 12% for all DOACs.

Conclusion: Minor, clinically insignificant, differences regarding centrifugation times were found in DOAC plasma concentrations. Importantly, no significant differences were found according to transportation modes for samples collected from patients. Although statistically significant differences were found depending on mode of transportation of spiked samples, relative bias was clinically acceptable. Thus, reduced centrifugation time and pneumatic tube transport should be considered to reduce turnaround time for rapid measurement of DOAC plasma concentrations.
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http://dx.doi.org/10.1111/ijlh.13729DOI Listing
October 2021

MicroRNA as Biomarkers for Platelet Function and Maturity in Patients with Cardiovascular Disease.

Thromb Haemost 2021 Jun 6. Epub 2021 Jun 6.

Department of Clinical Biochemistry, Thrombosis and Haemostasis Research Unit, Aarhus University Hospital, Aarhus, Denmark.

Patients with cardiovascular disease (CVD) are at increased risk of suffering myocardial infarction. Platelets are key players in thrombus formation and, therefore, antiplatelet therapy is crucial in the treatment and prevention of CVD. MicroRNAs (miRs) may hold the potential as biomarkers for platelet function and maturity. This systematic review was conducted using the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). To identify studies investigating the association between miRs and platelet function and maturity in patients with CVD, PubMed and Embase were searched on October 13 and December 13, 2020 without time boundaries. Risk of bias was evaluated using a standardized quality assessment tool. Of the 16 included studies, 6 studies were rated "good" and 10 studies were rated "fair." In total, 45 miRs correlated significantly with platelet function or maturity (rho ranging from -0.68 to 0.38, all  < 0.05) or differed significantly between patients with high platelet reactivity and patients with low platelet reactivity (-values ranging from 0.0001 to 0.05). Only four miRs were investigated in more than two studies, namely miR-223, miR-126, miR-21 and miR-150. Only one study reported on the association between miRs and platelet maturity. In conclusion, a total of 45 miRs were associated with platelet function or maturity in patients with CVD, with miR-223 and miR-126 being the most frequently investigated. However, the majority of the miRs were only investigated in one study. More data are needed on the potential use of miRs as biomarkers for platelet function and maturity in CVD patients.
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http://dx.doi.org/10.1055/s-0041-1730375DOI Listing
June 2021

Protein C deficiency; PROC gene variants in a Danish population.

Thromb Res 2020 01 30;185:153-159. Epub 2019 Nov 30.

Department of Clinical Biochemistry, Aarhus University Hospital, Denmark.

Introduction: Protein C deficiency is a heritable thrombophilia caused by numerous different genetic alterations in the protein C (PROC) gene. We aimed to identify variants causing protein C deficiency in a Danish population.

Material And Methods: Sanger sequencing of the PROC gene was performed in 20 probands and 26 relatives. In total, 30participants were previously diagnosed with protein C deficiency. Protein C activity was measured by a chromogenic substrate method (N = 40) and antigen level by an enzyme-linked immunosorbent assay (N = 26).

Results: Ten different single nucleotide variants were detected in 13 probands (65%) and in seven of the relatives previously diagnosed with protein C deficiency. Five variants were novel. The median protein C activity level was lower in participants with an identified variant (50% (range: 38-75%)) than in protein C deficient participants without a variant (65% (range: 36-73%); P = 0.18). A protein C activity of 57% resulted in the highest detection rate (12/13 (92%)). Likewise, the median antigen level was lower in participants with detectable variants than in participants without (49% (range: 35-99%) vs 70% (range: 41-101%); P = 0.09). No difference was found in venous thromboembolism (VTE) prevalence comparing participants with (12/20 (60%)) and without (7/10 (70%)) a variant (P = 0.59).

Conclusion: In a Danish population, a PROC gene variant was identified in 67% of participants previously diagnosed with protein C deficiency. Five variants were novel. The study confirmed an association between biochemical severity and the presence of a PROC gene variant. The VTE risk did not seem to differ between protein C deficient participants with and without a variant.
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http://dx.doi.org/10.1016/j.thromres.2019.11.027DOI Listing
January 2020

Pazopanib-Induced Liver Toxicity in Patients With Metastatic Renal Cell Carcinoma: Effect of UGT1A1 Polymorphism on Pazopanib Dose Reduction, Safety, and Patient Outcomes.

Clin Genitourin Cancer 2020 02 26;18(1):62-68.e2. Epub 2019 Sep 26.

Department of Oncology, Aarhus University Hospital, Aarhus, Denmark. Electronic address:

Background: Pazopanib can induce liver toxicity in patients with metastatic renal cell carcinoma (mRCC). We assessed the effect of a TA repeat polymorphism in the UGT1A1 (uridine diphosphate glucuronosyltransferase 1A1) gene encoding uridine diphosphate glucuronosyltransferase 1A1 on liver toxicity, dose reductions, and patient outcomes.

Patients And Methods: Patients with mRCC treated with first-line pazopanib developing liver toxicity underwent genotyping for the UGT1A1 polymorphism. Liver toxicity was assessed using the Common Terminology Criteria for Adverse Events, version 4.0. Progression-free survival and overall survival were assessed using the Kaplan-Meier and log-rank methods.

Results: Of 261 patients, 34 (13%) had developed liver toxicity after a median of 29 days (range, 5-155 days). Grade 4, 3, and 2 alanine aminotransferase or bilirubin had increased in 2 (6%), 17 (50%), and 8 (24%) patients, respectively. The UGT1A1 assessment demonstrated that 18 patients (53%) had TA6/TA7, 7 (21%) had TA7/TA7, and 9 (26%) had wild-type TA6/TA6. The UGT1A1 polymorphism was associated with improved median progression-free survival (TA6/TA6, 5.5 months; TA6/TA7, 34.2 months; TA7/TA7, 22.3 months; unknown UGT1A1 status, 9.2 months; UGT1A1 polymorphisms combined vs. unknown status, P = .021). UGT1A1 polymorphism was associated with improved median overall survival (TA6/TA6, 8.1 months, TA6/TA7 or TA7/TA7 not reached, unknown UGT1A1 status, 16.6 months; UGT1A1 polymorphisms combined vs. unknown status, P = .033). Patients with UGT1A1 polymorphism safely resumed pazopanib at ultra-low doses determined by the degree of liver toxicity and UGT1A1 polymorphism.

Conclusions: UGT1A1 polymorphisms were associated with improved outcomes, despite pazopanib interruption and dose reductions. UGT1A1 assessment could improve the management of pazopanib-induced liver toxicity in patients with mRCC.
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http://dx.doi.org/10.1016/j.clgc.2019.09.013DOI Listing
February 2020

Expanding the spectrum of genetic variants in the calcium-sensing receptor (CASR) gene in hypercalcemic individuals.

Clin Endocrinol (Oxf) 2019 11 9;91(5):683-690. Epub 2019 Sep 9.

Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Denmark.

Objective: Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominantly inherited disorder with overlapping biochemistry profile with primary hyperparathyroidism (PHPT), making the correct diagnosis a challenge. The objective of the study was to evaluate the results of the clinical work-up of a large group of hypercalcemic individuals.

Design: Cross-sectional study.

Patients: Patients undergoing clinical work-up of hypercalcemia.

Measurements: Molecular genetic analysis of the CASR gene and exon 2 of the AP2S1 gene. Plasma levels of ionized calcium and PTH as well as calcium creatinine clearance ratio (CCCR).

Results: A rare CASR variant was identified in 38 of 624 index patients (6.1%). A total of 18 CASR variants identified in this study were novel. No variants were identified in exon 2 of the AP2S1 gene. The majority of the variants (N = 16) were classified as likely pathogenic. The level of plasma calcium, plasma PTH and the CCCR was not affected by the type of variant (ie nonsense vs missense) (all P-values >.05). The CCCR was found to be significantly lower for variants in the transmembrane domain compared with variants located in the extracellular domain (P < .05). Plasma levels of calcium and PTH were not associated with the location of the variant (P > .05).

Conclusions: We expanded the spectrum of CASR variants in hypercalcemia with 18 novel variants, and suggest that the location of the CASR variant may affect calcium excretion as determined by the CCCR.
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http://dx.doi.org/10.1111/cen.14078DOI Listing
November 2019

Platelet microRNA expression and association with platelet maturity and function in patients with essential thrombocythemia.

Platelets 2020 26;31(3):365-372. Epub 2019 Jun 26.

Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.

Essential thrombocythemia (ET) is characterized by persistently elevated platelet counts and an increased risk of thromboembolic events. Dysregulated expression of small noncoding microRNAs (miRNAs) have been shown in ET and may influence platelet maturity and function in ET patients. In this study, we included 22 ET patients and 19 healthy controls to investigate the expression of 12 platelet miRNAs previously reported to be dysregulated in ET. Further, we investigated the correlation between the expression of selected miRNAs and platelet maturity and platelet function. Total RNA was isolated from platelets, and expression analyses were performed using TaqMan quantitative PCR (qPCR). Mean platelet volume (MPV) and immature platelet count and -fraction (IPC and IPF) were measured using the Sysmex XE-5000 automated haematology system. Platelet function was investigated by multiple electrode aggregometry (agonists: arachidonic acid (AA), thrombin-receptor-activating-peptide (TRAP) and adenosine diphosphate (ADP)), while platelet activation was determined by multi-colour flow cytometry (antibodies: bound-fibrinogen, CD63 and -selectin (CD62p), agonists: AA, TRAP and ADP). We showed that miR-9 and miR-490 were significantly upregulated in ET patients compared with healthy controls (-values < 0.01), while miR-10a, miR-28, miR-126, miR-155, miR-221, miR-222, miR-223 and miR-431 were significantly downregulated in ET patients (all -values < 0.001). A significant positive correlation was observed between miR-431 and MPV, IPC and IPF (all values < 0.05). The expression of miR-126 was negatively correlated with platelet aggregation induced by AA and TRAP ( < 0.05). In addition, we found the expression of miR-9 and miR-490 to be negatively correlated with the percentage of fibrinogen-, CD63- and -selectin- positive platelets using TRAP as agonist ( < 0.05). In conclusion, our data indicate that platelet microRNAs may play a role in ET and that specific microRNAs are correlated with platelet maturity and platelet function.
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http://dx.doi.org/10.1080/09537104.2019.1636019DOI Listing
September 2020

Whole blood platelet aggregation determined by the ROTEM platelet equipment; reference intervals and stability.

Platelets 2020 1;31(2):215-220. Epub 2019 Apr 1.

Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.

Point of care testing of residual effect of antiplatelet therapy in trauma patients or during major surgery may result in improved clinical management of significant bleeding. We included 121 healthy individuals (57 females and 64 males, aged 22-65 years) in order to establish reference intervals for platelet aggregation induced by adenosine diphosphate (ADPTEM, 10 µM), arachidonic acid (ARATEM, 0.42 mM) and thrombin activating peptide (TRAPTEM, 36 µM) employing the ROTEM platelet module. Further, the impact of citrate (3.2%) and hirudin (>15 µg/ml) as anticoagulants was evaluated. Finally, we investigated assay stability (15, 30, 60, and 120 min after blood sampling) (n = 8) and between-day variation (n = 5). We report reference intervals for 121 healthy individuals and reference intervals by gender. We observed significantly higher platelet aggregation in females than in males (all -values < 0.05). No correlation between age and platelet aggregation was observed, except for the parameter TRAPTEM amplitude (A6), in which a decline in A6 was observed with increasing age ( = 0.03). We observed significantly lower levels of platelet aggregation in citrate tubes than in hirudin tubes (all -values < 0.05), except from TRAPTEM maximum slope, where no significant difference was observed ( = 0.40).The stability was acceptable (≤20% deviation) for up to 120 min for ARATEM in citrate tubes, and up to 60 min for the ADPTEM and TRAPTEM assays in citrate tubes. In hirudin tubes we found ADPTEM and ARATEM assays to be stable for 60 min, while the stability of TRAPTEM in hirudin tubes was found to be stable for 30 min. Using citrate tubes, the between-day variation (mean coefficient of variation, CV) was 19-20% for ADPTEM, 19-26% for TRAPTEM, and 10% for ARATEM, whereas the mean CV was 11-13% for all three assays in hirudin tubes.In conclusion, we established combined and gender-specific reference intervals for three platelet aggregation assays in both citrate- and hirudin tubes. In citrate tubes, the stability of the ROTEM platelet assays was 60-120 min, while the stability in hirudin tubes was 30-60 min. The between-day variation was lowest for samples obtained in hirudin tubes.
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http://dx.doi.org/10.1080/09537104.2019.1595562DOI Listing
September 2020

SERPINC1 variants causing hereditary antithrombin deficiency in a Danish population.

Thromb Res 2019 Mar 31;175:68-75. Epub 2019 Jan 31.

Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark. Electronic address:

Introduction: Antithrombin deficiency is associated with increased risk of venous thromboembolism (VTE). We aimed to identify variants causing antithrombin deficiency in a Danish population.

Materials And Methods: We performed Sanger sequencing and, in relevant cases, multiplex ligation-dependent probe amplification analyses, in 46 individuals (23 index cases) with and 9 relatives without antithrombin deficiency. Furthermore, in order to explore whether a combination of antithrombin type II heparin binding site (HBS) deficiency and factor V Leiden single nucleotide variant (SNV) conferred a higher risk of VTE than either risk factor alone, we performed genotyping for factor V Leiden in most of the carriers of type II HBS deficiency (n = 25).

Results: We detected causal variants in all 46 carriers: three large and two small deletions, all causing type I antithrombin deficiency, and seven SNVs: one causing type I, one causing type II reactive site (RS), four causing type II HBS and one causing pleiotropic effect (PE) type II antithrombin deficiency. None of the relatives without antithrombin deficiency had the family variant. All detected SNVs have been reported previously. Majority (n = 27) of carriers had type II HBS deficiency, most often caused by the p.(Pro73Leu) SNV (n = 19). Heterozygosity for factor V Leiden was observed in three (3/25 = 12%) carriers of type II HBS deficiency. Only four (4/25 = 16%) carriers of type II HBS antithrombin deficiency experienced VTE, and two of these were heterozygous for factor V Leiden.

Conclusions: In a systematic search to identify variants causing hereditary antithrombin deficiency in a Danish population, we achieved a variant detection rate of 100%.
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http://dx.doi.org/10.1016/j.thromres.2019.01.022DOI Listing
March 2019

The clinical outcome of LMNA missense mutations can be associated with the amount of mutated protein in the nuclear envelope.

Eur J Heart Fail 2018 10 26;20(10):1404-1412. Epub 2018 Jun 26.

Department of Cardiology, Odense University Hospital, Odense, Denmark.

Aims: Lamin A/C mutations are generally believed to be associated with a severe prognosis. The aim of this study was to investigate disease expression in three affected families carrying different LMNA missense mutations. Furthermore, the potential molecular disease mechanisms of the mutations were investigated in fibroblasts obtained from mutation carriers.

Methods And Results: A LMNA-p.Arg216Cys missense mutation was identified in a large family with 36 mutation carriers. Disease expression was unusual with a late onset and a favourable prognosis. Two smaller families with severe disease expression were shown to carry a LMNA-p.Arg471Cys and LMNA-p.Arg471His mutation, respectively. LMNA gene and protein expression was investigated in eight different mutation carriers by quantitative reverse transcriptase polymerase chain reaction, Western blotting, immunohistochemistry, and protein mass spectrometry. The results showed that all mutation carriers incorporated mutated lamin protein into the nuclear envelope. Interestingly, the ratio of mutated to wild-type protein was only 30:70 in LMNA-p.Arg216Cys carriers with a favourable prognosis while LMNA-p.Arg471Cys and LMNA-p.Arg471His carriers with a more severe outcome expressed significantly more of the mutated protein by a ratio of 50:50.

Conclusion: The clinical findings indicated that some LMNA mutations may be associated with a favourable prognosis and a low risk of sudden death. Protein expression studies suggested that a severe outcome was associated with the expression of high amounts of mutated protein. These findings may prove to be helpful in counselling and risk assessment of LMNA families.
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http://dx.doi.org/10.1002/ejhf.1241DOI Listing
October 2018

Platelet characteristics in patients with essential thrombocytosis.

Cytometry B Clin Cytom 2018 11 3;94(6):918-927. Epub 2018 Sep 3.

Centre of Haemophilia and Thrombosis, Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.

Background: Essential thrombocytosis (ET) is a myeloproliferative disorder characterized by an increased platelet count. ET is associated with an increased risk of thrombosis, and procoagulant features of the disease may include an increased number of reactive reticulated platelets and an increased aggregation potential. We aimed to explore the association between platelet count, platelet turnover, and platelet aggregation in patients with ET.

Methods: We included 24 ET patients who discontinued antiplatelet therapy prior to blood sampling. Reticulated platelets were assessed as immature platelet count (IPC) and immature platelet fraction by automated flow cytometry (Sysmex XE-5000). Platelet aggregation was investigated by impedance aggregometry (Multiplate Analyzer) and aggregation potential by flow cytometry (NAVIOS).

Results: Our results showed that ET patients had increased IPC compared to healthy individuals (median 12.3 vs. median 6.9, P < 0.0001). Furthermore, a positive correlation between platelet count and impedance aggregation was demonstrated using arachidonic acid (r = 0.48, P = 0.02), thrombin-receptor-activating-peptide (r = 0.46, P = 0.03) and adenosine diphosphate (r = 0.56, P = 0.007) as agonists. Finally, an increased aggregation potential was demonstrated in ET patients compared to healthy individuals.

Conclusions: The study showed that ET patients compared to healthy individuals have an increased amount of reticulated platelets and increased aggregation potential. These findings might in part explain the increased thromboembolic risk in patients with ET. © 2018 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21642DOI Listing
November 2018

Pro-FHH: A Risk Equation to Facilitate the Diagnosis of Parathyroid-Related Hypercalcemia.

J Clin Endocrinol Metab 2018 07;103(7):2534-2542

Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Département de Physiologie, Paris, France.

Context: Parathyroid-related hypercalcemia is due to primary hyperparathyroidism (PHPT) or to familial hypocalciuric hypercalcemia (FHH). PHPT can lead to complications that necessitate parathyroidectomy. FHH is a rare genetic disease resembling PHPT; surgery is ineffective. A reliable method for distinguishing FHH from PHPT is needed.

Objective: To develop an easy-to-use tool to predict if a patient has PHPT.

Design: Retrospective analysis of two prospective cohorts. Development of an unsupervised risk equation (Pro-FHH).

Setting: University hospitals in Paris, France, and Aarhus, Denmark.

Participants: Patients (Paris: 65 with FHH, 85 with PHPT; Aarhus: 38 with FHH, 55 with PHPT) were adults with hypercalcemia and PTH concentration within normal range.

Main Outcome Measures: Performance of Pro-FHH to predict PHPT.

Results: Pro-FHH takes into account plasma calcium, PTH, and serum osteocalcin concentrations, and calcium-to-creatinine clearance ratio calculated from 24-hour urine collection (24h-CCCR). In the Paris cohort, area under the receiver operating characteristic curve (AUROC) of Pro-FHH was 0.961, higher than that of 24h-CCCR. With a cutoff value of 0.928, Pro-FHH had 100% specificity and 100% positive predictive value for the diagnosis of PHPT; it correctly categorized 51 of 85 patients with PHPT; the remaining 34 were recommended to undergo genetic testing. No patients with FHH were wrongly categorized. In an independent cohort from Aarhus, AUROC of Pro-FHH was 0.951, higher than that of 24h-CCCR.

Conclusion: Pro-FHH effectively predicted whether a patient has PHPT. A prospective trial is necessary to assess its usefulness in a larger population and in patients with elevated PTH concentration.
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http://dx.doi.org/10.1210/jc.2017-02773DOI Listing
July 2018

Lactase persistence genotyping on whole blood by loop-mediated isothermal amplification and melting curve analysis.

Clin Chim Acta 2018 Jul 26;482:50-56. Epub 2018 Mar 26.

Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark. Electronic address:

Background: The lactase persistence phenotype is controlled by a regulatory enhancer region upstream of the Lactase (LCT) gene. In northern Europe, specifically the -13910C > T variant has been associated with lactase persistence whereas other persistence variants, e.g. -13907C > G and -13915 T > G, have been identified in Africa and the Middle East. The aim of the present study was to compare a previously developed high resolution melting assay (HRM) with a novel method based on loop-mediated isothermal amplification and melting curve analysis (LAMP-MC) with both whole blood and DNA as input material.

Methods: To evaluate the LAMP-MC method, we used 100 whole blood samples and 93 DNA samples in a two tiered study. First, we studied the ability of the LAMP-MC method to produce specific melting curves for several variants of the LCT enhancer region. Next, we performed a blinded comparison between the LAMP-MC method and our existing HRM method with clinical samples of unknown genotype.

Results: The LAMP-MC method produced specific melting curves for the variants at position -13909, -13910, -13913 whereas the -13907C > G and -13915 T > G variants produced indistinguishable melting profiles.

Conclusion: The LAMP-MC assay is a simple method for lactase persistence genotyping and compares well with our existing HRM method.
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http://dx.doi.org/10.1016/j.cca.2018.03.029DOI Listing
July 2018

A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin-biased signaling.

Sci Signal 2018 02 20;11(518). Epub 2018 Feb 20.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, UK.

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that signals through G and G to stimulate cytosolic calcium (Ca) and mitogen-activated protein kinase (MAPK) signaling to control extracellular calcium homeostasis. Studies of loss- and gain-of-function mutations, which cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively, have revealed that the CaSR signals in a biased manner. Thus, some mutations associated with FHH1 lead to signaling predominantly through the MAPK pathway, whereas mutations associated with ADH1 preferentially enhance Ca responses. We report a previously unidentified ADH1-associated R680G CaSR mutation, which led to the identification of a CaSR structural motif that mediates biased signaling. Expressing CaSR in HEK 293 cells showed that this mutation increased MAPK signaling without altering Ca responses. Moreover, this gain of function in MAPK activity occurred independently of G and G and was mediated instead by a noncanonical pathway involving β-arrestin proteins. Homology modeling and mutagenesis studies showed that the R680G CaSR mutation selectively enhanced β-arrestin signaling by disrupting a salt bridge formed between Arg and Glu, which are located in CaSR transmembrane domain 3 and extracellular loop 2, respectively. Thus, our results demonstrate CaSR signaling through β-arrestin and the importance of the Arg-Glu salt bridge in mediating signaling bias.
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http://dx.doi.org/10.1126/scisignal.aan3714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166785PMC
February 2018

The impact of pneumatic tube transport on whole blood coagulation and platelet function assays.

Platelets 2018 Jun 14;29(4):421-424. Epub 2018 Feb 14.

a Department of Clinical Biochemistry , Aarhus University Hospital , Aarhus , Denmark.

Pneumatic tube is an attractive way to transport blood samples from the emergency department to the central laboratory facility. We aimed to investigate the impact of pneumatic tube transportation on blood samples for analysis of whole blood coagulation and platelet function. We included 21 healthy adult individuals and measured global coagulation assays by rotational thromboelastometry (ROTEM) and platelet aggregation induced by arachidonic acid (AA) and adenosine diphosphate (ADP) using impedance aggregometry (ROTEM Platelet), on samples transported manually or by pneumatic tube transport. Statistical testing was performed with paired tests with post-hoc Bonferroni correction for multiple testing. Our data revealed no difference in the far majority of ROTEM parameters (P > 0.003), while significantly decreased values were observed for INTEM clotting time (CT) (P = 0.002) and maximum clot firmness (MCF) including the amplitude after 10 min (A10) (P < 0.0001). No statistically significant difference was observed on impedance aggregometry results when manual transport was compared to pneumatic tube transport (P > 0.003). This study indicates that only minor and unsystematic differences between manual transport and pneumatic tube transport may be observed in ROTEM analyses, and that there is no influence from pneumatic tube transport on impedance aggregometry analyses using AA and ADP.
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http://dx.doi.org/10.1080/09537104.2018.1430361DOI Listing
June 2018

Platelet function investigation by flow cytometry: Sample volume, needle size, and reference intervals.

Platelets 2018 Mar 29;29(2):199-202. Epub 2017 Sep 29.

a Centre for Haemophilia and Thrombosis, Department of Clinical Biochemistry , Aarhus University Hospital , Aarhus , Denmark.

Flow cytometry is an increasingly used method for platelet function analysis because it has some important advantages compared with other platelet function tests. Flow cytometric platelet function analyses only require a small sample volume (3.5 mL); however, to expand the field of applications, e.g., for platelet function analysis in children, even smaller volumes are needed. Platelets are easily activated, and the size of the needle for blood sampling might be of importance for the pre-activation of the platelets. Moreover, to use flow cytometry for investigation of platelet function in clinical practice, a reference interval is warranted. The aims of this work were 1) to determine if small volumes of whole blood can be used without influencing the results, 2) to examine the pre-activation of platelets with respect to needle size, and 3) to establish reference intervals for flow cytometric platelet function assays. To examine the influence of sample volume, blood was collected from 20 healthy individuals in 1.0 mL, 1.8 mL, and 3.5 mL tubes. To examine the influence of the needle size on pre-activation, blood was drawn from another 13 healthy individuals with both a 19- and 21-gauge needle. For the reference interval study, 78 healthy adults were included. The flow cytometric analyses were performed on a NAVIOS flow cytometer (Beckman Coulter, Miami, Florida) investigating the following activation-dependent markers on the platelet surface; bound-fibrinogen, CD63, and P-selectin (CD62p) after activation with arachidonic acid, ristocetin, adenosine diphosphate, thrombin-receptor-activating-peptide, and collagen. The study showed that a blood volume as low as 1.0 mL can be used for platelet function analysis by flow cytometry and that both a 19- and 21-gauge needle can be used for blood sampling. In addition, reference intervals for platelet function analyses by flow cytometry were established.
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http://dx.doi.org/10.1080/09537104.2017.1353684DOI Listing
March 2018

Long-term Outcome of 4 Patients With Transcobalamin Deficiency Caused by 2 Novel TCN2 Mutations.

J Pediatr Hematol Oncol 2017 11;39(8):e430-e436

*Department of Pediatrics, King Abdullah International Medical Research Centre, Genetics Division #King Saud bin Abdulaziz University for Health Science, King Abdulaziz Medical City §Department of Pediatrics ∥Department of Medical Genetics, King Faisal Specialist Hospital and Research Centre ¶Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia †Department of Pediatrics, Genetics & Metabolism, University of Florida, Gainesville, FL ‡Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.

Cobalamin (vitamin B12 [Cbl]) is an essential cofactor for many biochemical pathways. Transcobalamin (TC) is required to internalize Cbl into the cells through membrane receptor-mediated endocytosis. Cbl is then processed in the cytoplasm and mitochondria by complementation factors leading to its active metabolites; methylcobalamin and 5-deoxyadenosyl-cobalamin. Deficiency of TC results in an elevation in methylmalonic acid and homocysteine. Patients usually present with macrocytic anemia, pancytopenia, failure to thrive, gastrointestinal symptoms, and neurological dysfunction. In this study, we report 4 patients from 2 unrelated families, with confirmed diagnosis of TC deficiency. Patients initially had a typical presentation of TC deficiency: severe diarrhea and vomiting, recurrent infections, stomatitis, macrocytic anemia, and neutropenia. Interestingly one of the patients was diagnosed at 3 months of age and developed ataxic gait related to cerebellar atrophy at the age of 14 months. His elder affected sibling was diagnosed at 5 months of age was completely normal. Two sibs, diagnosed at 2 months of age and immediately after birth, had autism spectrum disorder. Molecular investigations showed 2 novel mutations in TCN2 gene. Patients were treated and stayed stable on weekly injection of Cbl. In conclusion, TC deficiency has a wide heterogeneity in clinical phenotype, genotype, laboratory, and radiologic findings. Early detection of the disease and early initiation of aggressive parenteral treatment is probably associated with better prognosis and disease control.
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http://dx.doi.org/10.1097/MPH.0000000000000857DOI Listing
November 2017

AP2S1 and GNA11 mutations - not a common cause of familial hypocalciuric hypercalcemia.

Eur J Endocrinol 2017 Feb 15;176(2):177-185. Epub 2016 Nov 15.

Departments of Clinical Biochemistry

Objective: Familial hypocalciuric hypercalcemia (FHH) type 1 is caused by mutations in the gene encoding the calcium-sensing receptor (CASR). Recently, mutations affecting codon 15 in the gene AP2S1 have been shown to cause FHH type 3 in up to 26% of CASR-negative FHH patients. Similarly, mutations in the gene GNA11 have been shown to cause FHH type 2. We hypothesized that mutations in AP2S1 and GNA11 are causative in Danish patients with suspected FHH and that these mutations are not found in patients with primary hyperparathyroidism (PHPT), which is the main differential diagnostic disorder.

Design: Cross-sectional study.

Methods: We identified patients with unexplained hyperparathyroid hypercalcemia and a control group of verified PHPT patients through review of 421 patients tested for CASR mutations in the period 2006-2014. DNA sequencing of all amino acid coding exons including intron-exon boundaries in AP2S1 and GNA11 was performed.

Results: In 33 CASR-negative patients with suspected FHH, we found two (~6%) with a mutation in AP2S1 (p.Arg15Leu and p.Arg15His). Family screening confirmed the genotype-phenotype correlations. We did not identify any pathogenic mutations in GNA11. No pathogenic mutations were found in the PHPT control group.

Conclusions: We suggest that the best diagnostic approach to hyperparathyroid hypercalcemic patients suspected to have FHH is to screen the CASR and AP2S1 codon 15 for mutations. If the results are negative and there is still suspicion of an inherited condition (i.e. family history), then GNA11 should be examined.
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http://dx.doi.org/10.1530/EJE-16-0842DOI Listing
February 2017

Stability of direct oral anticoagulants in whole blood and plasma from patients in steady state treatment.

Thromb Res 2016 Dec 27;148:107-110. Epub 2016 Oct 27.

Department of Clinical Biochemistry, Aarhus University Hospital, Denmark; Institute of Clinical Medicine, Aarhus University, Aarhus, Denmark. Electronic address:

Using functional haemostasis assays, we demonstrated important differences in stability of direct oral anticoagulants (DOACs) in citrated whole blood and plasma from DOAC treated patients. Laboratories and clinicians should take this into consideration and adjust clinical practices accordingly.
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http://dx.doi.org/10.1016/j.thromres.2016.10.023DOI Listing
December 2016

Multiple endocrine neoplasia phenocopy revealed as a co-occurring neuroendocrine tumor and familial hypocalciuric hypercalcemia type 3.

Clin Case Rep 2016 Oct 18;4(10):922-927. Epub 2016 Aug 18.

Department of Endocrinology and Internal Medicine Aarhus University Hospital Aarhus Denmark.

Familial hypocalciuric hypercalcemia type 3 should be considered as differential diagnosis in patients with suspected primary hyperparathyroidism and/or suspected multiple neoplasia syndrome, as correct diagnosis will spare the patients for going through multiple futile parathyroidectomies and for the worry of being diagnosed with a cancer susceptibility syndrome.
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http://dx.doi.org/10.1002/ccr3.657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054464PMC
October 2016

The cardiovascular system in familial hypocalciuric hypercalcemia: a cross-sectional study on physiological effects of inactivating variants in the calcium-sensing receptor gene.

Eur J Endocrinol 2016 Oct 14;175(4):299-309. Epub 2016 Jul 14.

Department of Endocrinology and Internal MedicineAarhus University Hospital, Aarhus, Denmark Department of Clinical MedicineAarhus University, Aarhus, Denmark

Objective: Loss-of-function variants in the gene encoding the calcium-sensing receptor (CASR) result in familial hypocalciuric hypercalcemia (FHH), causing hypercalcemia with high normal or elevated parathyroid hormone levels. The CASR may also influence electrolyte and water homeostasis. It is unknown whether FHH affects cardiovascular health. We, therefore investigated whether FHH is associated with changes in the regulation of the cardiovascular system by measuring 24-h blood pressure (BP), arterial stiffness and vasoactive hormones.

Design: Cross-sectional study comparing 50 patients with FHH to age- and gender-matched controls.

Results: Studied subjects (69% women) had a mean age of 56years. A similar number of patients and controls (33%) were on treatment with antihypertensive drugs. Overall, no differences were found between groups in 24-h ambulatory BP or pulse wave velocity. However, compared with controls, diastolic BP during nighttime was lower in FHH females (60±5 vs 66±9mmHg, P<0.01) and higher in FHH males (69±6 vs 64±5mmHg, P=0.02). FHH was associated with a significantly higher plasma osmolality (P<0.01), higher plasma levels of vasopressin (P<0.01) and a higher renal excretion of epithelial sodium channels (ENaCs) (P=0.03), whereas urine aquaporin-2 and plasma sodium, aldosterone and renin did not differ between groups. FHH patients had a lower urinary volume with an increased osmolality if analyses were restricted to those not on treatments with antihypertensive drugs.

Conclusions: FHH does not seem to be associated with an increased risk of CVD.
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http://dx.doi.org/10.1530/EJE-16-0369DOI Listing
October 2016

False low holotranscobalamin levels in a patient with a novel TCN2 mutation.

Clin Chem Lab Med 2016 Nov;54(11):1739-1743

Background: Measurement of holotranscobalamin (holoTC) is increasingly used as a screening test for cobalamin (Cbl) deficiency. A level well below the reference interval strongly supports a deficient state. We examined a 21-year-old woman diagnosed as Cbl deficient because of an extremely low holoTC level as measured by the Abbott Architect Assay.

Methods: The patient was evaluated for Cbl deficiency employing an in-house holoTC method as well as other routine markers of Cbl status. Further analyses included exploration of the Cbl binding proteins employing gel filtration of a serum sample saturated with 57 Co-labeled Cbl and Sanger sequencing of exons 1-9 and the intron-exon boundaries of the TCN2 gene, the gene coding for transcobalamin (TC).

Results: The patient had normal hematological variables throughout. Despite initial treatment with Cbl, holoTC as measured by the Abbott assay remained low, while holoTC measured with the in-house assay was normal, and behaved as TC upon gel-filtration. By Sanger sequencing, we detected a homozygous single point mutation c.855T>A in exon 6 of TCN2, corresponding to a asparagine (Asn) to lysine (Lys) substitution in position 267 of the mature protein.

Conclusions: We describe a novel point mutation of the TCN2 gene. The mutation does not seem to interfere with the function of TC, but the mutation may well explain the low level of holoTC detected by the Abbott assay. Our results underscores that mutations of TCN2 have to be considered when implausible holoTC results are obtained.
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http://dx.doi.org/10.1515/cclm-2016-0063DOI Listing
November 2016

Investigation of platelet function and platelet disorders using flow cytometry.

Platelets 2016 22;27(1):66-74. Epub 2015 Apr 22.

a Department of Clinical Biochemistry , Centre for Haemophilia and Thrombosis, Aarhus University Hospital , Denmark and.

Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard-Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard-Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.
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http://dx.doi.org/10.3109/09537104.2015.1032919DOI Listing
October 2016

Genetic determinants of on-aspirin platelet reactivity: focus on the influence of PEAR1.

PLoS One 2014 31;9(10):e111816. Epub 2014 Oct 31.

Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.

Background: Platelet aggregation during aspirin treatment displays considerable inter-individual variability. A genetic etiology likely exists, but it remains unclear to what extent genetic polymorphisms determine platelet aggregation in aspirin-treated individuals.

Aim: To identify platelet-related single nucleotide polymorphisms (SNPs) influencing platelet aggregation during aspirin treatment. Furthermore, we explored to what extent changes in cyclooxygenase-1 activity and platelet activation may explain such influence.

Methods: We included 985 Danish patients with stable coronary artery disease treated with aspirin 75 mg/day mono antiplatelet therapy. Patients were genotyped for 16 common SNPs in platelet-related genes using standard PCR-based methods (TaqMan). Platelet aggregation was evaluated by whole blood platelet aggregometry employing Multiplate Analyzer (agonists: arachidonic acid and collagen) and VerifyNow Aspirin. Serum thromboxane B2 was measured to confirm aspirin adherence and was used as a marker of cyclooxygenase-1 activity. Soluble P-selectin was used as marker of platelet activation. Platelet aggregation, cyclooxygenase-1 activity, and platelet activation were compared across genotypes in adjusted analyses.

Results: The A-allele of the rs12041331 SNP in the platelet endothelial aggregation receptor-1 (PEAR1) gene was associated with reduced platelet aggregation and increased platelet activation, but not with cyclooxygenase-1 activity. Platelet aggregation was unaffected by the other SNPs analyzed.

Conclusion: A common genetic variant in PEAR1 (rs12041331) reproducibly influenced platelet aggregation in aspirin-treated patients with coronary artery disease. The exact biological mechanism remains elusive, but the effect of this polymorphism may be related to changes in platelet activation. Furthermore, 14 SNPs previously suggested to influence aspirin efficacy were not associated with on-aspirin platelet aggregation.

Clinical Trial Registration: ClinicalTrials.gov NCT01383304.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111816PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4216141PMC
December 2015

Activating calcium-sensing receptor gene variants in children: a case study of infant hypocalcaemia and literature review.

Acta Paediatr 2014 Nov 24;103(11):1117-25. Epub 2014 Aug 24.

Department of Paediatrics, Aarhus University Hospital, Aarhus, Denmark.

Unlabelled: Autosomal dominant hypocalcaemia (ADH) is caused by activating variants in the calcium-sensing receptor (CASR) gene, but detailed information on the paediatric phenotype is limited. The current paper presents a case of severe ADH and systematically reviews the literature on ADH in children.

Conclusion: We found that the severity of clinical neurological symptoms was inversely related to serum calcium levels and a high prevalence of renal calcifications and/or basal ganglia calcifications in children with ADH.
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http://dx.doi.org/10.1111/apa.12743DOI Listing
November 2014

Increased trabecular volumetric bone mass density in Familial Hypocalciuric Hypercalcemia (FHH) type 1: a cross-sectional study.

Calcif Tissue Int 2014 Aug 4;95(2):141-52. Epub 2014 Jun 4.

Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Tage-Hansens Gade 2, 8000, Aarhus C, Denmark.

Familial Hypocalciuric Hypercalcaemia (FHH) Type 1 is caused by an inactivating mutation in the calcium-sensing receptor (CASR) gene resulting in elevated plasma calcium levels. We investigated whether FHH is associated with change in bone density and structure. We compared 50 FHH patients with age- and gender-matched population-based controls (mean age 56 years, 69 % females). We assessed areal BMD (aBMD) by DXA-scans and total, cortical, and trabecular volumetric BMD (vBMD) as well as bone geometry by quantitative computed tomography (QCT) and High-Resolution peripheral-QCT (HR-pQCT). Compared with controls, FHH females had a higher total and trabecular hip vBMD and a lower cortical vBMD and hip bone volume. Areal BMD and HRpQCT indices did not differ except an increased trabecular thickness and an increased vBMD at the transition zone between cancellous and cortical bone in of the tibia in FHH. Finite element analyses showed no differences in bone strength. Multiple regression analyses revealed correlations between vBMD and P-Ca(2+) levels but not with P-PTH. Overall, bone health does not seem to be impaired in patients with FHH. In FHH females, bone volume is decreased, with a lower trabecular volume but a higher vBMD, whereas cortical vBMD is decreased in the hip. This may be due to either an impaired endosteal resorption or corticalization of trabecular bone. The smaller total bone volume suggests an impaired periosteal accrual, but bone strength is not impaired. The findings of more pronounced changes in females may suggest an interaction between sex hormones and the activity of the CaSR on bone.
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http://dx.doi.org/10.1007/s00223-014-9877-0DOI Listing
August 2014

Genotyping increases the yield of angiotensin-converting enzyme in sarcoidosis--a systematic review.

Dan Med J 2014 May;61(5):A4815

Lungemedicinsk Afdeling LUB, Hjertecentret, Aarhus Universitetshospital, Nørrebrogade 44, 8000 Aarhus C, Denmark.

Introduction: The diagnosis of sarcoidosis is challenging and involves radiological, clinical and paraclinical evaluation, the latter including the measurement of serum angiotensin-converting enzyme activity (s-ACE), which is elevated in about 60% of sarcoidosis patients. The normal inter-individual biological variation of s-ACE is large. Approximately 50% of the variation is due to a genomic insertion/deletion (I/D) polymorphism in the ACE gene.

Methods: We searched the MEDLINE library for articles presenting genotype-based reference intervals for s-ACE in healthy people. We summarised the results as weighted mean DD/II ratios of s-ACE. We also summarised the presented frequencies of the genotypes.

Results: We identified nine studies presenting genotype-based reference intervals. All studies found a significant difference between mean s-ACE in the three genotype groups DD, ID and II. The mean DD/II ratio was 1.85 (range: 1.79-1.92) for all studies, 2.01 (1.92-2.10) for Caucasians and 1.64 (1.55-1.73) for Asians. The median frequencies of genotypes among Caucasians were 23% II, 45% ID and 30% DD, and 45% II, 49% ID and 14% DD among Asians.

Conclusion: Genotyping for the I/D polymorphism increases the benefit of s-ACE since all studies found significantly different levels between genotype groups in healthy subjects. Genotyping is of special value if s-ACE is between the upper 97.5 percentile for genotype II and DD since values in this interval are at risk of being misclassified. Due to assay variation, genotype-specific reference levels should be verified locally.
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May 2014

Truncating plakophilin-2 mutations in arrhythmogenic cardiomyopathy are associated with protein haploinsufficiency in both myocardium and epidermis.

Circ Cardiovasc Genet 2014 Jun 4;7(3):230-40. Epub 2014 Apr 4.

From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).

Background: Arrhythmogenic cardiomyopathy (AC) is a hereditary cardiac condition associated with ventricular arrhythmias, heart failure, and sudden death. The disease is most often caused by mutations in the desmosomal gene for plakophilin-2 (PKP2), which is expressed in both myocardial and epidermal tissue. This study aimed to investigate protein expression in myocardial tissue of patients with AC carrying PKP2 mutations and elucidate whether keratinocytes of the same individuals exhibited a similar pattern of protein expression.

Methods And Results: Direct sequencing of 5 AC genes in 71 unrelated patients with AC identified 10 different PKP2 mutations in 12 index patients. One patient, heterozygous for a PKP2 nonsense mutation, developed severe heart failure and underwent cardiac transplantation. Western blotting and immunohistochemistry of the explanted heart showed a significant decrease in PKP2 protein expression without detectable amounts of truncated PKP2 protein. Cultured keratinocytes of the patient showed a similar reduction in PKP2 protein expression. Nine additional PKP2 mutations were investigated in both cultured keratinocytes and endomyocardial biopsies from affected individuals. It was evident that PKP2 mutations introducing a premature termination codon in the reading frame were associated with PKP2 transcript and protein levels reduced to ≈50%, whereas a missense variant did not seem to affect the amount of PKP2 protein.

Conclusions: The results of this study showed that truncating PKP2 mutations in AC are associated with low expression of the mutant allele and that the myocardial protein expression of PKP2 is mirrored in keratinocytes. These findings indicate that PKP2 haploinsufficiency contributes to pathogenesis in AC.
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http://dx.doi.org/10.1161/CIRCGENETICS.113.000338DOI Listing
June 2014

Development of a high-resolution melting genotyping assay for the angiotensin I converting enzyme insertion/deletion polymorphism and establishment of genotype-specific reference intervals in a Danish population.

Ann Clin Biochem 2015 Jan 2;52(Pt 1):105-12. Epub 2014 Apr 2.

Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.

Background: The serum-angiotensin I converting enzyme (s-ACE) activity is influenced by a genetic insertion/deletion (I/D) polymorphism in the ACE gene, and the resulting large interindividual variation in s-ACE limits the use of normal reference intervals in the evaluation of sarcoidosis. In this study, we developed a new method for genotyping the I/D polymorphism in ACE and established genotype-specific reference intervals in order to improve the diagnostic accuracy and the value for treatment of sarcoidosis.

Methods: The new genotyping assay is based on high-resolution melting (HRM) using LCGreen + and was used to genotype 400 healthy Danish individuals. The assay was compared to a real-time polymerase chain reaction (RT-PCR) assay in a validation set of 86 samples. Enzyme activity in serum was measured using the Infinity™ ACE Liquid Stable Reagent from Thermo adapted for the ABX Pentra analyzer.

Results: There was full concordance between genotyping assays. The three genotypes II, ID and DD were present with a frequency of 0.23, 0.51 and 0.26. The distribution of s-ACE values in the total population was non-Gaussian (non-parametric 95% reference interval 12.0-60.0 U/L). The median activities of the genotypes differed significantly (P<0.0001). Ninety-five per cent non-parametric reference intervals for the subpopulations were determined to 6.3-38.5, 14.0-56.0 and 23.3-71.2 U/L for II, ID and DD, respectively.

Conclusion: We have developed a simple and robust method for ACE genotyping and determined genotype-specific reference intervals for s-ACE concentrations in the Danish population. The new reference intervals may increase the value of s-ACE measurements.
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http://dx.doi.org/10.1177/0004563214529261DOI Listing
January 2015

The LMNA mutation p.Arg321Ter associated with dilated cardiomyopathy leads to reduced expression and a skewed ratio of lamin A and lamin C proteins.

Exp Cell Res 2013 Nov 31;319(19):3010-9. Epub 2013 Aug 31.

Research Unit for Molecular Medicine, Aarhus University and Aarhus University Hospital, Aarhus, Denmark.

Dilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by cardiac chamber enlargement and reduced systolic function of the left ventricle. Mutations in the LMNA gene represent the most frequent known genetic cause of DCM associated with disease of the conduction systems. The LMNA gene generates two major transcripts encoding the nuclear lamina major components lamin A and lamin C by alternative splicing. Both haploinsuffiency and dominant negative effects have been proposed as disease mechanism for premature termination codon (PTC) mutations in LMNA. These mechanisms however are still not clearly established. In this study, we used a representative LMNA nonsense mutation, p.Arg321Ter, to shed light on the molecular disease mechanisms. Cultured fibroblasts from three DCM patients carrying this mutation were analyzed. Quantitative reverse transcriptase PCR and sequencing of these PCR products indicated that transcripts from the mutant allele were degraded by the nonsense-mediated mRNA decay (NMD) mechanism. The fact that no truncated mutant protein was detectable in western blot (WB) analysis strengthens the notion that the mutant transcript is efficiently degraded. Furthermore, WB analysis showed that the expression of lamin C protein was reduced by the expected approximately 50%. Clearly decreased lamin A and lamin C levels were also observed by immunofluorescence microscopy analysis. However, results from both WB and nano-liquid chromatography/mass spectrometry demonstrated that the levels of lamin A protein were more reduced suggesting an effect on expression of lamin A from the wild type allele. PCR analysis of the ratio of lamin A to lamin C transcripts showed unchanged relative amounts of lamin A transcript suggesting that the effect on the wild type allele was operative at the protein level. Immunofluorescence microscopy analysis showed no abnormal nuclear morphology of patient fibroblast cells. Based on these data, we propose that heterozygosity for the nonsense mutation causes NMD degradation of the mutant transcripts blocking expression of the truncated mutant protein and an additional trans effect on lamin A protein levels expressed from the wild type allele. We discuss the possibility that skewing of the lamin A to lamin C ratio may contribute to ensuing processes that destabilize cardiomyocytes and trigger cardiomyopathy.
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http://dx.doi.org/10.1016/j.yexcr.2013.08.024DOI Listing
November 2013

Muscle function and quality of life are not impaired in familial hypocalciuric hypercalcemia: a cross-sectional study on physiological effects of inactivating variants in the calcium-sensing receptor gene (CASR).

Eur J Endocrinol 2013 Sep 28;169(3):349-57. Epub 2013 Aug 28.

Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Tage-Hansens Gade 2, 8000 Aarhus C, Denmark.

Background: Familial hypocalciuric hypercalcemia (FHH) is often due to inactivating variants in the calcium-sensing receptor (CASR) gene causing chronically elevated plasma calcium levels with inappropriately normal or elevated parathyroid hormone levels. In patients with primary hyperparathyroidism, the state of hyperparathyroid hypercalcemia is associated with reduced muscle strength and impaired quality of life (QoL).

Objective: To study whether FHH affects muscle function, postural stability, and QoL.

Design: In a cross-sectional study, we investigated muscle strength (handgrip, elbow flexion/extension, and knee flexion/extension), balance function, physical activity, and QoL in 50 patients with FHH and in a similar number of age- and gender-matched population-based healthy controls. All but one of the FHH cases had genetically verified inactivating variants in the CASR gene.

Results: Studied subjects (n=100, 68% females) had a mean age of 56.0 years. Muscle strength as assessed by measuring maximum force and maximum force production did not differ between the groups. Neither did groups differ in terms of QoL, physical activity, or postural stability, as assessed during normal standing with eyes open, normal standing with eyes closed, semi-tandem standing, or tandem standing. Adjustment for vitamin D status (plasma 25-hydroxyvitamin D levels) and BMI did not change results.

Conclusion: Despite a state of chronic hypercalcemia, muscle strength, balance function, and QoL are not impaired in patients with FHH. Our findings are reassuring for patients with FHH as they should not be considered as having a severe disease.
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http://dx.doi.org/10.1530/EJE-13-0224DOI Listing
September 2013
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