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
•We study disease mechanisms in DCM patients carrying PTC mutations in the LMNA gene.•The mutant transcript is degraded by the nonsense mediated mRNA decay system.•Skewed lamin A to lamin C protein ratio expressed from the wild type allele.•We suggest a combined pathomechanism: haploinsuffiency plus lamin A/C imbalance.
Antithrombin deficiency is associated with increased risk of venous thromboembolism (VTE). We aimed to identify variants causing antithrombin deficiency in a Danish population.
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).
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.
In a systematic search to identify variants causing hereditary antithrombin deficiency in a Danish population, we achieved a variant detection rate of 100%.
•Systematic report of SERPINC1 variants causing antithrombin deficiency in a Danish population•Variant detection rate was 100%.•p.(Pro73Leu), a type II HBS single nucleotide variant, was observed in 41% of all carriers.•The prevalence of type II HBS deficiency in the general population might be underestimated.
Newly produced immature platelets are larger, contain higher amounts of residual RNA, and are more reactive than mature platelets. Flow cytometry using the SYTO-13 dye is a method for the subdivision ...of immature platelets from mature platelets based on the labelling of intracellular platelet RNA, enabling the simultaneous investigation of the reactivity of each platelet population. This method provides detailed information on several aspects of platelet physiology using a combination of platelet surface markers and agonists. Currently, no standardized protocol exists across laboratories. Here, we describe a flow cytometry protocol in detail to investigate platelet reactivity and its relation to platelet maturity. We analyzed 20 healthy individuals with the protocol and compared the platelet subpopulation with the highest SYTO-13 labelling (in the first quintile, "SYTO-high") corresponding to the most immature platelets (highest RNA content) with the platelet subpopulation with the lowest SYTO-13 labelling (in the fifth quintile, "SYTO-low") corresponding to the mature platelets with the lowest RNA content. SYTO-high platelets had overall significantly increased platelet reactivity compared with that of SYTO-low platelets. The presented method may be a valuable research tool for the analysis of platelet reactivity and its relation to platelet maturity.
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.
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).
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.
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.
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.
When screening for α‐thalassemia in children, adult cut‐offs for mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) are generally applied to guide genetic evaluation. However, the ...normal ranges for MCV and MCH are lower in children than in adults, so we hypothesized that using age‐matched cut‐offs could lead to a more rational diagnostic strategy. The aim of this study was to evaluate if age‐matched cut‐offs could be applied advantageously. Data on children referred to a hemoglobin fractionation at the Department of Clinical Biochemistry, Aarhus University Hospital between 2016–2021 were identified in the laboratory information system. α‐globin gene (HBA1/HBA2) genotyping was performed using multiplex gap‐polymerase chain reaction. A total of 387 children were identified. HBA1/HBA2‐genotyping was performed in 207 children (53%), and α‐thalassemia was diagnosed in 47 children (23%) with −α3.7/αα being the predominant genotype (13%). We found that 23 children had MCV and MCH levels in the normal age‐matched range, and two of these children (9%) were α+ thalassemia carriers with three functional α‐globin genes. Using age‐specific cut‐off levels resulted in a reduction of 23 (11%) genotypes performed. In conclusion, applying age‐matched cut‐offs for MCV and MCH when screening children for α‐thalassemia lead to 11% fewer genotypes performed while 9% carriers of α+ thalassemia (of the medically innocuous genotype −α3.7/αα) would have been overlooked.
Abstract 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.
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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