Background Disrupted pre-mRNA splicing is a frequent deleterious mechanism in hereditary cancer. We aimed to functionally analyze candidate spliceogenic variants of the breast cancer susceptibility... Show moreBackground Disrupted pre-mRNA splicing is a frequent deleterious mechanism in hereditary cancer. We aimed to functionally analyze candidate spliceogenic variants of the breast cancer susceptibility gene CHEK2 by splicing reporter minigenes.Methods A total of 128 CHEK2 splice-site variants identified in the Breast Cancer After Diagnostic Gene Sequencing (BRIDGES) project (https://cordis.europa.eu/project/id/634935) were analyzed with MaxEntScan and subsetted to 52 variants predicted to impact splicing. Three CHEK2 minigenes, which span all 15 exons, were constructed and validated. The 52 selected variants were then genetically engineered into the minigenes and assayed in MCF-7 (human breast adenocarcinoma) cells.Results Of 52 variants, 46 (88.5%) impaired splicing. Some of them led to complex splicing patterns with up to 11 different transcripts. Thirty-four variants induced splicing anomalies without any trace or negligible amounts of the full-length transcript. A total of 89 different transcripts were annotated, which derived from different events: single- or multi-exon skipping, alternative site-usage, mutually exclusive exon inclusion, intron retention or combinations of the abovementioned events. Fifty-nine transcripts were predicted to introduce premature termination codons, 7 kept the original open-reading frame, 5 removed the translation start codon, 6 affected the 5 & PRIME;UTR (Untranslated Region), and 2 included missense variations. Analysis of variant c.684-2A > G revealed the activation of a non-canonical TG-acceptor site and exon 6 sequences critical for its recognition.Conclusions Incorporation of minigene read-outs into an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme allowed us to classify 32 CHEK2 variants (27 pathogenic/likely pathogenic and 5 likely benign). However, 20 variants (38%) remained of uncertain significance, reflecting in part the complex splicing patterns of this gene. Show less
Background We explored the potential of emerging and conventional urinary kidney injury biomarkers in recipients of living donor (LD) or donation after circulatory death (DCD) kidney... Show moreBackground We explored the potential of emerging and conventional urinary kidney injury biomarkers in recipients of living donor (LD) or donation after circulatory death (DCD) kidney transplantation, patients with chronic kidney disease (CKD), and individuals from the general population. Methods Urine samples from kidney allograft recipients with mild (LD; n = 199) or severe (DCD; n = 71) ischemia-reperfusion injury (IRI) were analyzed for neutrophil gelatinase-associated lipocalin (NGAL), insulin-like growth factor-binding protein 7 (IGFBP7), tissue inhibitor of metalloproteinases 2 (TIMP2), kidney injury molecule-1 (KIM-1), chemokine C-X-C motif (CXCL9), solute carrier family 22 member 2 (SLC22A2), nephrin, and uromodulin (UMOD) by quantitative multiplex LC-MS/MS analysis. The fold-change in biomarker levels was determined in mild and severe IRI and in patients with CKD stage 1-2 (n = 127) or stage & GE;3 (n = 132) in comparison to the general population (n = 1438). Relationships between the biomarkers and total protein, & beta;2-microglobulin (B2M), creatinine, and osmolality were assessed. Results NGAL, IGFBP7, TIMP2, KIM-1, CXCL9, and UMOD were quantifiable, whereas nephrin and SLC22A2 were below the limit of detection. Kidney injury biomarkers were increased up to 6.2-fold in allograft recipients with mild IRI and 8.3-fold in recipients with severe IRI, compared to the reference population, with the strongest response observed for NGAL and B2M. In CKD stage 1-2, B2M, NGAL, IGFBP7, TIMP2, KIM-1, UMOD, and CXCL9 were not altered, but in individuals with CKD stage & GE;3, B2M, NGAL, and KIM-1 were increased up to 1.3-fold. IGFBP7, TIMP2, NGAL, and CXCL9 were strongly correlated (all r & GE; 0.8); correlations with B2M and TP were smaller (all r & LE; 0.6). Conclusions IRI, but not stable CKD, was associated with increased urinary levels of kidney injury biomarkers determined by LC-MS/MS. Absolute and multiplexed protein quantitation by LC-MS/MS is an effective strategy for biomarker panel evaluation for translation toward the clinical laboratory. Show less
Background: Medical results generated by European CE Marking for In Vitro Diagnostic or in-house tests should be traceable to higher order reference measurement systems (RMS), such as International... Show moreBackground: Medical results generated by European CE Marking for In Vitro Diagnostic or in-house tests should be traceable to higher order reference measurement systems (RMS), such as International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-endorsed reference measurement procedures (RMPs) and reference materials. Currently, serum apolipoprotein (a) [apo(a)] is recognized as a novel risk factor for cardiovascular risk assessment and patient management. The former RMS for serum apo(a) is no longer available; consequently, an International System of Units (SI)-traceable, ideally multiplexed, and sustainable RMS for apo(a) is needed. Methods: A mass spectrometry (MS)-based candidate RMP (cRMP) for apo(a) was developed using quantitative bottom-up proteomics targeting 3 proteotypic peptides. The method was provisionally validated according to ISO 15193 using a single human serum based calibrator traceable to the former WHO-IFCC RMS. Results: The quantitation of serum apo(a) was by design independent of its size polymorphism, was linear from 3.8 to 456 nmol/L, and had a lower limit of quantitation for apo(a) of 3.8 nmol/L using peptide LFLEPTQADIALLK. Interpeptide agreement showed Pearson Rs of 0.987 and 0.984 for peptides GISSTVTGR and TPENYPNAGLTR, and method comparison indicated good correspondence (slopes 0.977, 1.033, and 1.085 for LFLEPTQADIALLK, GISSTVTGR, and TPENYPNAGLTR). Average within-laboratory imprecision of the cRMP was 8.9%, 11.9%, and 12.8% for the 3 peptides. Conclusions: A robust, antibody-independent, MS-based cRMP was developed as higher order RMP and an essential part of the apo(a) traceability chain and future RMS. The cRMP fulfils predefined analytical performance specifications, making it a promising RMP candidate in an SI-traceable MS-based RMS for apo(a). Show less
Dikaios, I.; Althaus, H.; Angles-Cano, E.; Ceglarek, U.; Coassin, S.; Cobbaert, C.M.; ... ; IFCC Working Grp Apolipoprot Mass 2023
Background: Elevated concentrations of lipoprotein(a) [Lp(a)] are directly related to an increased risk of cardiovascular diseases, making it a relevant biomarker for clinical risk assessment.... Show moreBackground: Elevated concentrations of lipoprotein(a) [Lp(a)] are directly related to an increased risk of cardiovascular diseases, making it a relevant biomarker for clinical risk assessment. However, the lack of global standardization of current Lp(a) measurement procedures (MPs) leads to inconsistent patient care. The International Federation for Clinical Chemistry and Laboratory Medicine working group on quantitating apolipoproteins by mass spectrometry (MS) aims to develop a next-generation SI (International system of units)-traceable reference measurement system consisting of a MS-based, peptide-calibrated reference measurement procedure (RMP) and secondary serum-based reference materials (RMs) certified for their apolipoprotein(a) [apo(a)] content. To reach measurement standardization through this new measurement system, 2 essential requirements need to be fulfilled: a sufficient correlation among the MPs and appropriate commutability of future serum-based RMs. Methods: The correlation among the candidate RMP (cRMP) and immunoassay-based MPs was assessed by measuring a panel of 39 clinical samples (CS). In addition, the commutability of 14 different candidate RMs was investigated. Results: Results of the immunoassay-based MPs and the cRMPs demonstrated good linear correlations for the CS but some significant sample-specific differences were also observed. The results of the commutability study show that RMs based on unspiked human serum pools can be commutable with CS, whereas human pools spiked with recombinant apo(a) show different behavior compared to CS. Conclusions: The results of this study show that unspiked human serum pools are the preferred candidate secondary RMs in the future SI-traceable Lp(a) Reference Measurement System. Show less
Current dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum... Show moreCurrent dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum apolipoproteins A-I, (a), B, C-I, C-II, C-III, and E may give better pathophysiological insight into the root causes of dyslipidemia. To facilitate the future adoption of clinical serum apolipoprotein (apo) profiling for precision medicine, strategies for accurate testing should be developed in advance.Recent discoveries in basic science and translational medicine set the stage for the IFCC Working Group on Apolipoproteins by Mass Spectrometry. Main drivers were the convergence of unmet clinical needs in cardiovascular disease (CVD) patients with enabling technology and metrology. First, the residual cardiovascular risk after accounting for established risk factors demonstrates that the current lipid panel is too limited to capture the full complexity of lipid metabolism in patients. Second, there is a need for accurate test results in highly polymorphic and atherogenic apolipoproteins such as apo(a). Third, sufficient robustness of mass spectrometry technology allows reproducible protein quantification at the molecular level. Fourth, several calibration hierarchies in the revised ISO 17511:2020 guideline facilitate metrological traceability of test results, the highest achievable standard being traceability to SI.This article outlines the conceptual approach aimed at achieving a novel, multiplexed Reference Measurement System (RMS) for seven apolipoproteins based on isotope dilution mass spectrometry and peptide-based calibration. This RMS should enable standardization of existing and emerging apolipoprotein assays to SI, within allowable limits of measurement uncertainty, through a sustainable network of Reference Laboratories. Show less
BACKGROUND: Originally studied as a mechanism to understand eclampsia-related deaths during pregnancy, fetal cells in maternal blood have more recently garnered attention as a noninvasive source of... Show moreBACKGROUND: Originally studied as a mechanism to understand eclampsia-related deaths during pregnancy, fetal cells in maternal blood have more recently garnered attention as a noninvasive source of fetal material for prenatal testing. In the 21st century, however, intact fetal cells have been largely supplanted by circulating cell-free placental DNA for aneuploidy screening. Instead, interest has pivoted to the ways in which fetal cells influence maternal biology. In parallel, an increasing appreciation of the consequences of maternal cells in the developing fetus has occurred.CONTENT: In this review, we highlight the potential clinical applications and functional consequences of the bidirectional trafficking of intact cells between a pregnant woman and her fetus. Fetal cells play a potential role in the pathogenesis of maternal disease and tissue repair. Maternal cells play an essential role in educating the fetal immune system and as a factor in transplant acceptance. Naturally occurring maternal microchimerism is also being explored as a source of hematopoietic stem cells for transplant in fetal hematopoietic disorders.SUMMARY: Future investigations in humans need to include complete pregnancy histories to understand maternal health and transplant success or failure. Animal models are useful to understand the mechanisms underlying fetal wound healing and/or repair associated with maternal injury and inflammation. The lifelong consequences of the exchange of cells between a mother and her child are profound and have many applications in development, health, and disease. This intricate exchange of genetically foreign cells creates a permanent connection that contributes to the survival of both individuals. Show less
BACKGROUND: Noninvasive trisomy 21 detection performed by use of massively parallel sequencing is achievable with high diagnostic sensitivity and low false-positive rates. Detection of fetal... Show moreBACKGROUND: Noninvasive trisomy 21 detection performed by use of massively parallel sequencing is achievable with high diagnostic sensitivity and low false-positive rates. Detection of fetal trisomy 18 and 13 has been reported as well but seems to be less accurate with the use of this approach. The reduced accuracy can be explained by PCR-introduced guanine-cytosine (GC) bias influencing sequencing data. Previously, we demonstrated that sequence data generated by single molecule sequencing show virtually no GC bias and result in a more pronounced noninvasive detection of fetal trisomy 21. In this study, single molecule sequencing was used for noninvasive detection of trisomy 18 and 13.METHODS: Single molecule sequencing was performed on the Helicos platform with free DNA isolated from maternal plasma from 11 weeks of gestation onward (n = 17). Relative sequence tag density ratios were calculated against male control plasma samples and results were compared to those of previous karyotyping.RESULTS: All trisomy 18 fetuses were identified correctly with a diagnostic sensitivity and specificity of 100%. However, low diagnostic sensitivity and specificity were observed for fetal trisomy 13 detection.CONCLUSIONS: We successfully applied single molecule sequencing in combination with relative sequence tag density calculations for noninvasive trisomy 18 detection using free DNA from maternal plasma. However, noninvasive trisomy 13 detection was not accurate and seemed to be influenced by more than just GC content. Show less
BACKGROUND: The Ca2+-dependent protein phosphatase enzyme calcineurin (Cn) (protein phosphatase 3) is best known for its role as director of the adaptive immune response. One of its principal... Show moreBACKGROUND: The Ca2+-dependent protein phosphatase enzyme calcineurin (Cn) (protein phosphatase 3) is best known for its role as director of the adaptive immune response. One of its principal substrates is the nuclear factor of activated T cells (NFAT), which translocates to the nucleus after dephosphorylation to mediate gene transcription. Drugs targeting Cn (the Cn inhibitors tacrolimus and cyclosporin A) have revolutionized posttransplantation therapy in allograft recipients by considerably reducing rejection rates. CONTENT: Owing primarily to intensive study of the side effects of the Cn inhibitors, the unique importance of Cn and Cn/NFAT signaling in the normal physiological processes of many other cell and tissue types is becoming more evident. During the last decade, it has become clear that an extensive and diverse array of clinical conditions can be traced back, at least in part, to a disturbed Cn-signaling axis. Hence, both diagnostics and therapeutic monitoring could benefit from a technique that conveniently reads out Cn/NFAT operative status. SUMMARY: This review outlines the current knowledge on the pathologic conditions that have calcineurin as a common denominator and reports on the progress that has been made toward successfully applying Cn and Cn/NFAT activity markers in molecular diagnostics. Show less
